WO2013126834A1 - Profils d'expression de micro-arn œsophagiens dans œsophagite éosinophile - Google Patents

Profils d'expression de micro-arn œsophagiens dans œsophagite éosinophile Download PDF

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WO2013126834A1
WO2013126834A1 PCT/US2013/027503 US2013027503W WO2013126834A1 WO 2013126834 A1 WO2013126834 A1 WO 2013126834A1 US 2013027503 W US2013027503 W US 2013027503W WO 2013126834 A1 WO2013126834 A1 WO 2013126834A1
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mirnas
hsa
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Marc E. Rothenberg
Thomas Xuefeng LU
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Children's Hospital Medical Center
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Priority to EP13751420.4A priority Critical patent/EP2817417B1/fr
Priority to CA2865154A priority patent/CA2865154A1/fr
Priority to AU2013222129A priority patent/AU2013222129B2/en
Priority to US14/380,672 priority patent/US9260756B2/en
Publication of WO2013126834A1 publication Critical patent/WO2013126834A1/fr
Priority to US14/989,243 priority patent/US9624545B2/en

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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the invention disclosed herein generally relates to diagnosis, treatment, and/or management of eosinophilic esophagitis, asthma, and/or allergic diseases, disorders, and/or conditions arising therefrom and/or related thereto.
  • Eosinophilic esophagitis (EE, also abbreviated EoE in some publications) is an emerging worldwide disease characterized by marked eosinophil infiltration of the esophageal mucosal epithelium (>15 eosinophils/ high power field (hpf)) that is refractory to acid suppressive therapy and is associated with chronic symptoms from childhood into adulthood (see, e.g., Furuta, G. et al. Gastroenterology 133: 1342-63 (2007); Assa'ad, A. et al. J Allergy Clin. Immunol. 119:731-8 (2007); Straumann, A. and Simon, H.
  • EE symptoms mimic gastroesophageal reflux disease (GERD) and can vary with age.
  • Patients with EE can have gastrointestinal complaints that typically include, but are not limited to, failure to thrive, vomiting, abdominal pain, dysphagia, and food impactions (see, e.g., Furuta, G. et al. Gastroenterology 133: 1342-63 (2007); Liacouras, C. et al. J Pediatr. Gastroenterol. Nutr. 45:370-91 (2007)).
  • EE diagnosis involves endoscopy, which is an invasive and inconvenient procedure. The endoscopy procedure is then commonly followed by biopsy analysis. EE provides an opportunity to closely study human inflammatory diseases, as obtaining tissue specimens via endoscopy is routine standard-of-care, and the biopsy material is amenable to molecular analysis (see, e.g., Liacouras, C. et al. J. Allergy Clin. Immunol. 128:3-20 (2011); Abonia, J. et al. J. Allergy Clin. Immunol. 126: 140-9 (2010)).
  • Embodiments of the invention encompass methods of treating a patient with eosinophilic esophagitis (EE), including obtaining a sample from a patient, analyzing the sample to determine a level of one or more miRNAs associated with EE, determining whether the level of the one or more miRNAs is up-regulated or down-regulated relative to a level of the one or more miRNAs measured in a normal individual, wherein the presence of an elevated or reduced level of one or more miRNAs associated with EE results in the patient being diagnosed with EE, and treating the patient with an appropriate therapeutic strategy based upon the diagnosis.
  • EE eosinophilic esophagitis
  • the one or more miRNAs associated with EE can include, for example, miR-886-5p, miR-886-3p, miR-222*, miR-7, miR-29b, miR-642, miR-339-5p, miR-21, miR-21 *, miR-142-5p, miR-146a, miR-146b, miR-142-3p, miR-132, miR-212, miR-592, miR-92a-l *, miR-223*, miR-223, miR-801, miR-106b*, miR- 375, miR-211, miR-210, miR-365, miR-203, miR-193a-5p, miR-193b, miR-193a-3p, let-7c, miR-144*, or miR-30a-3p.
  • the one or more miRNAs associated with EE can include, for example, miR-21, miR-223, miR-375, miR-142-3p, miR-146a, or miR- 146b. In some embodiments, the one or more miRNAs associated with EE can include, for example, miR-21, miR-223, or miR-375.
  • the determination of whether the level(s) of the one or more miRNAs associated with EE are elevated or reduced relative to a level of the one or more miRNAs measured in a normal individual can be combined with a determination of a level(s) of one or more additional biomarkers associated with EE.
  • the one or more additional biomarkers associated with EE can include, for example, an mRNA biomarker.
  • the one or more additional biomarkers associated with EE can include, for example, eotaxin-3.
  • the sample can be, for example, an esophageal tissue sample.
  • the sample can be, for example, a plasma or serum sample.
  • the sample can be, for example, a buccal sample, an oral swish, or saliva.
  • the appropriate therapeutic strategy for a patient diagnosed with EE can include, for example, allergen removal, steroid treatment, dietary management, proton pump inhibitor (PPI) therapy, administration of one or more topical glucocorticoids, humanized antibodies against one or more relevant cytokines and/or mediators, one or more small molecule inhibitors of an eosinophil and/or allergic disease activation pathway, one or more small molecule inhibitors capable of modulating miRNA levels and/or as severing as stem-loop processing inhibitors, and/or any combination thereof.
  • the topical glucocorticoid can include, for example, fluticasone, budesonide, and/or ciclesonide.
  • the humanized antibody against a relevant cytokine or mediator can include, for example, an antibody against eotaxin-1, eotaxin-3, IL-13, IL-5, IL-5Ra, CD49D, SIGLEC-8, IgE, CD300A, TSLP, and/or IL-33.
  • the small molecule inhibitor can include, for example, a notch-signaling inhibitor or an inhibitor or antagonist of CCR3, CCL11, VLA4, CRTH2, prostaglandin D2, histamine H4 receptor, IL-13, IL-4, and/or the common ⁇ chain.
  • the appropriate therapeutic strategy includes using any of the one or more miRNA(s) associated with EE found to be elevated relative to the level(s) of the one or more miRNAs measured in a normal individual or using one or more corresponding modified miRNA(s) as a therapeutic target or agent.
  • the appropriate therapeutic strategy includes administering to the patient one or more agents such as, for example, an anti-miRNA oligonucleotide (antagomir), an antisense oligonucleotide, a locked nucleic acid, an RNA competitive inhibitor or decoy, and/or a viral vector expressing one or more miRNA genes, and the like.
  • agents such as, for example, an anti-miRNA oligonucleotide (antagomir), an antisense oligonucleotide, a locked nucleic acid, an RNA competitive inhibitor or decoy, and/or a viral vector expressing one or more miRNA genes, and the like.
  • the antagomir can be directed against a miRNA found to be elevated relative to the level(s) of the one or more miRNAs measured in a normal individual.
  • the antagomir against the up- regulated miRNA can be, for example, a miR-21, miR-223, miR-146a, and/or miR-146b antagomir, and the like.
  • the competitive inhibitor against the elevated miRNA can be, for example, an IGF1 or IGF1R inhibitor, and the like.
  • the IGF1R inhibitor can be, for example, NVP-AEW541 and/or pricopodophyllin, and the like.
  • the viral vector expressing one or more miRNA genes can be, for example, a lentiviral vector, an adenoviral vector, and/or an adeno-associated virus, and the like.
  • Some embodiments of the methods further include a determination of eosinophilic esophagitis or chronic esophagitis, wherein the presence of a non-elevated or non-reduced level of one or more miRNAs associated with eosinophilic esophagitis results in the patient being diagnosed with chronic esophagitis.
  • the appropriate therapeutic strategy can include, for example, antacid administration, H2 agonist administration, and/or PPI therapy.
  • Some embodiments of the methods further include a determination of active eosinophilic esophagitis or eosinophilic esophagitis in remission, wherein the one or more miRNAs can include, for example, miR-886-5p, miR-886-3p, miR-222*, miR-7, miR- 29b, miR-642, miR-339-5p, miR-21, miR-21 *, miR-142-5p, miR-146a, miR-146b, miR-142- 3p, miR-132, miR-212, miR-592, miR-92a-l *, miR-223*, miR-223, miR-801, miR-106b*, miR-375, miR-211, miR-210, miR-365, miR-203, miR-193a-5p, miR-193b, miR-193a-3p, let- 7c, miR-144*, and miR-30a-3p
  • Some embodiments of the methods further include a determination of EE disease severity, wherein a highly up-regulated or highly down-regulated level of the one or more miRNAs relative to the level(s) of the one or more miRNAs measured in a normal individual is indicative of a severe case of EE.
  • a patient diagnosed with EE can be determined to be compliant with and/or exposed to steroid treatment, wherein an elevated level of miR-675 following treatment indicates that the patient is compliant with and/or exposed to steroid treatment.
  • a patient diagnosed with EE and treated with a steroid can be determined to be responsive or non-responsive to steroid treatment, wherein an elevated level of miR-675 following treatment indicates that the patient is responsive to steroid treatment.
  • a patient diagnosed with EE can be determined to be likely to be responsive or non-responsive to anti-IL-13 treatment, wherein an elevated level of one or more miRNAs associated with periostin levels indicates that the patient is likely to be responsive to anti-IL-13 treatment.
  • the one or more miRNAs associated with periostin levels can include, for example, miR-223 and/or miR-375.
  • Embodiments of the invention are also directed to methods of diagnosing a patient with eosinophilic esophagitis (EE), the methods including obtaining a sample from a patient, analyzing the sample to determine a level of one or more miRNAs associated with EE in adult patients, and determining whether the level of the one or more miRNAs are up- regulated or down-regulated relative to a level of the one or more miRNAs measured in a normal individual, wherein the presence of an elevated or reduced level of one or more miRNAs associated with EE results in the patient being diagnosed with EE.
  • EE eosinophilic esophagitis
  • Embodiments of the invention are also directed to methods of treating a patient with an eosinophilic disorder, the methods including, obtaining a sample from a patient, analyzing the sample to determine a level of one or more miRNAs associated with an eosinophilic disorder, determining whether the level of the one or more miRNAs is up- regulated or down-regulated relative to a level of the one or more miRNAs measured in a normal individual, wherein the presence of an elevated or reduced level of one or more miRNAs associated with an eosinophilic disorder results in the patient being diagnosed with an eosinophilic disorder, and treating the patient with an appropriate therapeutic strategy based upon the diagnosis.
  • the eosinophilic disorder can be, for example, an eosinophilic gastrointestinal disorder (EGID).
  • EGID eosinophilic gastrointestinal disorder
  • the eosinophilic disorder can be, for example, asthma.
  • the one or more miRNAs associated with asthma can be, for example, miR-375.
  • the sample can include, for example, lung and/or lung epithelial cells.
  • Embodiments of the invention are also directed to a diagnostic kit, test, or array, including materials for quantification of at least two analytes, wherein the at least two analytes are miRNAs associated with eosinophilic esophagitis (EE).
  • EE eosinophilic esophagitis
  • the at least two analytes can include, for example, miR-21, miR-223, and miR-375.
  • the at least two analytes can include, for example, miR-886-5p, miR-886-3p, miR-222*, miR-7, miR-29b, miR-642, miR-339-5p, miR-21, miR-21 *, miR-142-5p, miR-146a, miR-146b, miR-142-3p, miR-132, miR-212, miR- 592, miR-92a-l *, miR-223*, miR-223, miR-801, miR-106b*, miR-375, miR-211, miR-210, miR-365, miR-203, miR-193a-5p, miR-193b, miR-193a-3p, let-7c, miR-144*, and miR-30a- 3p.
  • the at least two analytes can include, for example, miR-21, miR- 223, miR-375, miR-142-3p, miR-146a, and miR-146b. In some embodiments, the at least two analytes can include, for example, miR-21, miR-223, miR-375, miR-142-3p, miR-146a, and miR-146b.
  • the at least two analytes can include all of miR-21, miR-223, and miR-375. In some embodiments, the at least two analytes can include all of miR-21, miR-223, miR-375, miR-142-3p, miR-146a, and miR-146b.
  • the at least two analytes can include all of miR-886-5p, miR-886-3p, miR-222*, miR-7, miR- 29b, miR-642, miR-339-5p, miR-21, miR-21 *, miR-142-5p, miR-146a, miR-146b, miR-142- 3p, miR-132, miR-212, miR-592, miR-92a-l *, miR-223*, miR-223, miR-801, miR-106b*, miR-375, miR-211, miR-210, miR-365, miR-203, miR-193a-5p, miR-193b, miR-193a-3p, let- 7c, miR-144*, and miR-30a-3p.
  • the diagnostic kit, test, or array can include a gene chip.
  • the gene chip includes a low density array.
  • the diagnostic kit, test, or array can include a surface with a DNA array.
  • Figure 1 depicts microRNA (miRNA) expression profiles in normal patients and EE patients. The figure presents a heatmap of 21 up-regulated and 11 down- regulated miRNAs in EE patients compared to normal controls.
  • Figures 2A-F depict quantitative real time polymerase chain reaction (qRT- PCR) verification of a selected set of differentially expressed miRNAs in normal patients and EE patients.
  • Figures 3 A-C depict miRNA expression profiles in normal patients and EE patients compared to chronic esophagitis patients and EE patients responding to glucocorticoid therapy, as well as correlation of miR-21 and miR-223 with EE signature genes.
  • Figure 3 A presents a heatmap showing the expression levels of 32 differentially expressed miRNAs in EE patients compared to chronic esophagitis patients and EE patients in remission after glucocorticoid therapy.
  • Figure 3B illustrates the correlation of EE signature genes with miR- 21.
  • Figure 3C illustrates the correlation of EE signature genes with miR-223. The significance of the correlation was plotted as the negative log of p value for each gene.
  • Figures 4A-B depict the miR As that are differentially regulated in EE patients in remission.
  • Figure 4 A presents a heatmap showing the expression level of miR-675, which is the only miRNA differentially regulated in EE patients that responded to glucocorticoid therapy compared to normal controls, chronic esophagitis patients, and EE patients.
  • Figures 5A-B depict regions identified as primary transcripts for miR-21 and miR-223 (pri-miR-21 and pri-miR-223) during RNA sequencing (RNA-Seq) analysis.
  • Figure 5 A illustrates the identification of the pri-miR-21 region in the RNA-Seq analysis.
  • the normalized coverage tracks for the mRNA-seq are displayed, along with the spliced reads that are present in exons of VMP1.
  • the lack of spliced reads present between the regions outside of the exons of VMP1 indicates that it is a portion of pri-miR-21.
  • FIG. 5B illustrates the identification of the pri-miR-223 region in the RNA-Seq analysis.
  • the normalized coverage tracks for the mRNA-seq are displayed, but there are no reads spanning junctions because there are no splicing events present in this region.
  • the regions annotated as "exon” are not necessarily true exons of a gene but regions that showed significant expression patterns similar to that of a gene's exons. They were annotated as such to facilitate the identification of the region to use for the correlation analysis.
  • FIGS. 6A-C depict gene enrichment analyses of miR-21 and miR-223 co- regulated genes in EE patients, with extensive enrichment of genes with functional features associated with T cell polarization, IFNy signaling, and regulation of eosinophilia among genes whose expression is correlated with miR-21 and miR-223 expression in the esophageal biopsies.
  • the networks are shown as Cytoscape (open source software, see http ⁇ colon slash slash> www ⁇ dot> cytoscape ⁇ dot> org) graph networks generated from ToppCluster (Cincinnati Children's Hospital Medical Center, Cincinnati, OH) network analysis.
  • Figure 6A illustrates the abstracted interactions between miR-21 and miR-223 co-regulated target genes.
  • Figure 6B illustrates the miR-21 targets that are significantly correlated with miR-21 expression or in the EE transcriptome; targets are shown as hexagons.
  • miR-21 co-regulated targets are significantly enriched in genes that regulate interleukin production.
  • Figure 6C illustrates the Pearson correlation of miR-21 and IL-12p35 expression levels in esophageal biopsies from EE patients.
  • FIG. 7 depicts gene enrichment analysis of miR-21 and miR-223 co- regulated genes in EE patients.
  • the miR-223 targets that are significantly correlated with miR-223 expression or in the EE transcriptome are highlighted as hexagons.
  • Figures 8A-C depict miRNAs differentially expressed in the plasma of active EE patients and EE remission patients compared to normal controls.
  • the relative expression levels were normalized to miR-16.
  • N 13-14 plasma samples per group; data are represented as mean ⁇ SEM; NS: not significant with P > .05.
  • FIG. 10 depicts esophageal eosinophil counts for wild type (WT) mice and miR-21 gene knockout (KO) mice under saline control (Sal) or upon exposure to the allergen Aspergillus fumigatus (Asp).
  • Figures 11A-B depict miR-21 induction during eosinophil differentiation.
  • Figure 11A illustrates the purity of cultured eosinophils at day 14; eosinophils are identified as CCR3 + Siglec-F + cells.
  • Figure 11B illustrates miR-21 expression levels during the eosinophil differentiation culture, as determined by quantitative polymerase chain reaction (qPCR) normalized to U6.
  • qPCR quantitative polymerase chain reaction
  • Figures 12A-C depict the growth of eosinophil progenitor cells from miR- 2 ⁇ ⁇ ' ⁇ mice and miR-21 +/+ controls during the ex vivo eosinophil culture.
  • Figure 12A illustrates the total cell number of eosinophil cultures;
  • Figure 12B illustrates the total number of neutrophil cultures derived from miR-21 +/+ and miR-2r /_ mice.
  • N 6 per group; data are represented as mean ⁇ SEM.
  • Figure 12C displays the morphology of miR-21 +/+ and miR-2r /_ cultured eosinophils at day 12, as determined by Diff-Quik (Fisher Scientific, Pittsburgh, PA) staining.
  • Figure 13 depicts levels of apoptosis in the eosinophil progenitor culture from the miR-21 +/+ and miR-2r /_ mice.
  • Levels of annexin V and 7AAD staining during eosinophil differentiation culture were determined by fluorescence-activated cell sorting (FACS).
  • the viable cells are annexin V " / 7AAD " .
  • the early apoptotic cells are annexin V + / 7AAD "
  • the late apoptotic cells are annexin V + / 7AAD + .
  • Figures 14A-C depict blood eosinophil percentage and bone marrow eosinophil colony forming unit capacity in the miR-21 +/+ and miR-21 "7" mice.
  • Figure 14B illustrates bone marrow eosinophil colony forming unit (CFU-Eos), and
  • Figure 14C illustrates neutrophil colony forming unit (CFU-G) capacity from miR-21 +/+ and miR-21 "7” mice.
  • N 4 per group; data are represented as mean ⁇ SEM.
  • Figures 15A-D present heatmaps of differentially regulated genes between miR-21 +/+ and miR-21 "7" eosinophil progenitor cultures at day 8 and day 12.
  • Figure 15A presents a heatmap of differentially regulated genes at day 8 of the eosinophil differentiation culture.
  • Figure 15B presents a heatmap of differentially regulated genes at day 12 of the eosinophil differentiation culture.
  • Figure 15C depicts the quantitative RT-PCR verification of a selected set of differentially expressed genes between miR-21 +/+ and miR-21 "7" eosinophil progenitor cultures.
  • Figure 15D illustrates a functional enrichment analysis of differentially regulated genes in the eosinophil progenitor cultures at day 12.
  • the networks are shown as Cytoscape (open source software) graph networks generated from ToppCluster (Cincinnati Children's Hospital Medical Center) network analysis.
  • Figure 16 depicts a biological function enrichment analysis of differentially regulated genes in eosinophil progenitor cultures at day 8. The figure illustrates an analysis of the most significant biological functions represented by the differentially regulated genes between miR-21 +/+ and miR-21 "7" eosinophil progenitor cultures.
  • Figures 17A-C depict miR-223 induction during eosinophil differentiation.
  • Figure 17A presents a schematic of the ex vivo bone marrow-derived eosinophil culture.
  • Figure 17B illustrates the purity of cultured eosinophils after 14 days; eosinophils are identified as CCR3 + Siglec-F + cells.
  • Figure 17C illustrates miR-223 expression levels during the eosinophil differentiation culture.
  • N 3 per group; data are represented as mean ⁇ SEM.
  • Figures 18A-C depict the growth of eosinophil progenitor cells and morphology of mature eosinophils from miR-223 +/+ and miR-223 "7" cultures during the ex vivo eosinophil differentiation culture.
  • Figure 18B illustrates the morphology of miR-223 +/+ and miR-223 "7" eosinophil progenitor culture at day 8, 10, and 12, as determined by modified Giemsa (Diff-Quik) staining.
  • Figure 18C illustrates the morphology of miR-223 +/+ and miR-223 "7” cultured eosinophils at day 14, as determined by Diff-Quik (Fisher Scientific) staining.
  • FIG. 19 depicts levels of IGF1R during eosinophil differentiation culture from the miR-223 +/+ and miR-223 "7" mice.
  • the figure presents a western blot showing levels of pre-IGFIR and IGF1R in eosinophil differentiation cultures derived from miR-223 +/+ and miR-223 "7” mice from day 4 to day 14.
  • the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control.
  • Figures 20A-C depict the finding that the increased growth observed in cultures derived from miR-223 "7" mice can be reversed by an IGF1R inhibitor.
  • Figure 20A displays data for bone marrow-derived eosinophils from miR-223 +/+ and miR-223 "7" mice; eosinophils were treated with 2 ⁇ picropodophyllin (PPP, an IGF1R inhibitor) or an equivalent volume of dimethyl sulfoxide (DMSO). Growth was measured by cell counting using a hemacytometer.
  • Figure 20B presents a western blot showing levels of IGF1R expression in miR-223 +/+ and miR-223 "7” cells after 2-day treatment with PPP.
  • Figures 21A-B depict the increased growth observed in eosinophil progenitor cultures derived from miR-223 "7" mice, coupled with a delay in eosinophil progenitor differentiation.
  • Figure 21A displays CCR3 expression at day 8, day 10, and day 12 of the eosinophil progenitor culture in miR-223 "7” cultures compared to miR-223 +/+ cultures, as measured by qPCR normalized to HPRT1.
  • N 3 per group; data are represented as mean ⁇ SEM.
  • Figure 2 IB displays levels of CCR3 expression during eosinophil differentiation culture, as determined by FACS staining of surface CCR3 and Siglec F levels; mature eosinophils are identified as CCR3 + Siglec-F + cells.
  • Figures 22A-B depict the mature eosinophil levels in the blood and eosinophil progenitor levels in the bone marrow of miR-223 +/+ and miR-223 "7" mice.
  • Figures 23 A-D present heatmaps of differentially regulated genes between miR-223 +/+ and miR-223 "7" eosinophil progenitor cultures at day 8 and day 12, along with their most strongly associated biological functions.
  • Figure 22 A presents a heatmap of differentially regulated genes at day 8 of the eosinophil differentiation culture.
  • Figure 22B illustrates a functional enrichment analysis of differentially regulated genes in the eosinophil progenitor cultures at day 8. The networks are shown as Cytoscape (open source software) graph networks generated from ToppCluster (Cincinnati Children's Hospital Medical Center) network analysis.
  • Figure 22C presents a heatmap of differentially regulated genes at day 12 of the eosinophil differentiation culture.
  • Figure 22D illustrates an analysis of the most significant biological functions represented by the differentially regulated genes between miR- 223 +/+ and miR-223 "7" eosinophil progenitor cultures at day 12.
  • Figure 24 depicts a biological function enrichment analysis of differentially regulated genes in eosinophil progenitor cultures at day 8. The figure illustrates an analysis of the most significant biological functions represented by the differentially regulated genes between miR-223 +/+ and miR-223 "7" eosinophil progenitor cultures.
  • Figures 25A-B depict the miRNA expression profile in human esophageal epithelial cells and human bronchial epithelial cells after 24 hours of IL-13 stimulation.
  • Figure 25 A presents a heatmap of 4 down-regulated and 2 up-regulated miRNAs in IL-13- stimulated human esophageal epithelial cells compared to controls.
  • Figure 25B presents a heatmap of 4 down-regulated and 2 up-regulated miRNAs in IL-13 -stimulated human bronchial epithelial cells compared to controls.
  • FIGS 26A-D depict qRT-PCR verification of miR-375 expression in IL- 13 -stimulated human esophageal epithelial cells and human bronchial epithelial cells.
  • Expression of miR-375 was determined in ( Figure 26 A) IL-13 -stimulated human esophageal epithelial cells compared to controls and (Figure 26B) IL-13 -stimulated human bronchial epithelial cells compared to controls.
  • Figure 26C displays a kinetic analysis of miR-375 expression in IL-13 -stimulated esophageal epithelial cells.
  • Figure 27 depicts expression of miR-375 in a doxycycline-induced IL-13 lung transgenic experimental asthma model.
  • the relative expression levels of miR-375 were determined by qPCR normalized to U6.
  • N 6 mice per group; data are represented as mean ⁇ SEM.
  • Figures 28A-C depict expression of miR-375 in esophageal biopsies from EE patients and the correlation with esophageal eosinophil counts and EE signature genes.
  • Figure 28B displays the correlation between miR-375 expression and esophageal eosinophil counts.
  • Figure 28C displays the correlation between miR-375 expression and EE signature genes. The significance of the correlation was plotted as the negative log of p value for each gene. The dashed line represents significance level after false discovery rate correction.
  • Figure 29 depicts miR-375 expression levels in different cell types.
  • the relative expression level of miR-375 in different cell types was determined by qPCR normalized to U6.
  • N 3 per group; data are represented as mean ⁇ SEM.
  • Figures 30A-B depict genes differentially regulated by miR-375 in esophageal epithelial cells before and after IL-13 stimulation.
  • Figure 30A presents a heatmap showing genes differentially expressed in esophageal epithelial cell line transduced with either a control vector or a pre-miR-375 expression vector, before and after IL-13 stimulation.
  • Figure 30B displays a functional enrichment analysis of pathways affected by miR-375 under IL-13 -stimulated conditions.
  • the networks are shown as Cytoscape (open source software) graph networks generated from ToppCluster (Cincinnati Children's Hospital Medical Center) network analysis.
  • Figure 31 depicts a biological function enrichment analysis of all miR-375- regulated genes. The figure illustrates an analysis of the most significant diseases and disorders represented by all genes differentially regulated by miR-375.
  • Figure 32 depicts the expression levels of thymic stromal lymphopoietin (TSLP) in polyinosinic:polycytidylic acid (poly(LC)) stimulated pre-miR-375 -transduced TE- 7 esophageal epithelial cells compared to controls.
  • Control-transduced and pre-miR-375- transduced TE-7 cells were stimulated with 25 ⁇ g/mL poly(LC) for 0, 2, 4, and 8 hours.
  • Expression levels were determined by qPCR normalized to HPRT1.
  • N 4 per group; data are represented as mean ⁇ SEM.
  • a subject refers to any member of the animal kingdom. In some embodiments, a subject is a human patient.
  • sample encompasses a sample obtained from a subject or patient.
  • the sample can be of any biological tissue or fluid.
  • samples include, but are not limited to, sputum, saliva, buccal sample, oral sample, blood, serum, mucus, plasma, urine, blood cells (e.g., white cells), circulating cells (e.g. stem cells or endothelial cells in the blood), tissue, core or fine needle biopsy samples, cell-containing body fluids, free floating nucleic acids, urine, stool, peritoneal fluid, and pleural fluid, liquor cerebrospinalis, tear fluid, or cells therefrom.
  • Samples can also include sections of tissues such as frozen or fixed sections taken for histological purposes or microdissected cells or extracellular parts thereof.
  • a sample to be analyzed can be tissue material from a tissue biopsy obtained by aspiration or punch, excision or by any other surgical method leading to biopsy or resected cellular material.
  • Such a sample can comprise cells obtained from a subject or patient.
  • the sample is a body fluid that include, for example, blood fluids, serum, mucus, plasma, lymph, ascitic fluids, gynecological fluids, or urine but not limited to these fluids.
  • the sample can be a saline swish, a buccal scrape, a buccal swab, and the like.
  • blood can include, for example, plasma, serum, whole blood, blood lysates, and the like.
  • assessing includes any form of measurement, and includes determining if an element is present or not.
  • determining includes determining if an element is present or not.
  • evaluating includes determining if an element is present or not.
  • assessing includes determining if an element is present or not.
  • determining includes determining if an element is present or not.
  • evaluating includes determining if an element is present or not.
  • assessing includes determining if an element is present or not.
  • assaying can be used interchangeably and can include quantitative and/or qualitative determinations.
  • diagnosis or monitoring with reference to a disease state or condition refers to a method or process of determining if a subject has or does not have a particular disease state or condition or determining the severity or degree of the particular disease state or condition.
  • treatment refers to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or can be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a subject, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease and/or relieving one or more disease symptoms.
  • Treatment can also encompass delivery of an agent or administration of a therapy in order to provide for a pharmacologic effect, even in the absence of a disease or condition.
  • treatment is used in some embodiments to refer to administration of a compound of the present invention to mitigate a disease or a disorder in a host, preferably in a mammalian subject, more preferably in humans.
  • treatment can include includes: preventing a disorder from occurring in a host, particularly when the host is predisposed to acquiring the disease, but has not yet been diagnosed with the disease; inhibiting the disorder; and/or alleviating or reversing the disorder.
  • the term “prevent” does not require that the disease state be completely thwarted (see Webster's Ninth Collegiate Dictionary).
  • the term preventing refers to the ability of the skilled artisan to identify a population that is susceptible to disorders, such that administration of the compounds of the present invention can occur prior to onset of a disease. The term does not mean that the disease state must be completely avoided.
  • modulated or modulation can refer to both up regulation (i.e., activation or stimulation, e.g., by agonizing or potentiating) and down regulation (i.e., inhibition or suppression, e.g., by antagonizing, decreasing or inhibiting), unless otherwise specified or clear from the context of a specific usage.
  • up regulation i.e., activation or stimulation, e.g., by agonizing or potentiating
  • down regulation i.e., inhibition or suppression, e.g., by antagonizing, decreasing or inhibiting
  • the term "marker” or “biomarker” refers to a biological molecule, such as, for example, a nucleic acid, peptide, protein, hormone, and the like, whose presence or concentration can be detected and correlated with a known condition, such as a disease state. It can also be used to refer to a differentially expressed gene whose expression pattern can be utilized as part of a predictive, prognostic or diagnostic process in healthy conditions or a disease state, or which, alternatively, can be used in methods for identifying a useful treatment or prevention therapy.
  • the term “expression levels” refers, for example, to a determined level of biomarker expression.
  • pattern of expression levels refers to a determined level of biomarker expression compared either to a reference (e.g. a housekeeping gene or inversely regulated genes, or other reference biomarker) or to a computed average expression value (e.g. in DNA-chip analyses).
  • a pattern is not limited to the comparison of two biomarkers but is more related to multiple comparisons of biomarkers to reference biomarkers or samples.
  • a certain “pattern of expression levels” can also result and be determined by comparison and measurement of several biomarkers as disclosed herein and display the relative abundance of these transcripts to each other.
  • a "reference pattern of expression levels” refers to any pattern of expression levels that can be used for the comparison to another pattern of expression levels.
  • a reference pattern of expression levels is, for example, an average pattern of expression levels observed in a group of healthy or diseased individuals, serving as a reference group.
  • Eosinophilic esophagitis (EE, also referred to as EoE in some publications) is a condition characterized by elevated esophageal levels of eosinophils. EE is considered to be a T H 2-associated disease (see, e.g., Blanchard, C. et al. J. Allergy Clin. Immunol.
  • EE diagnosis requires endoscopy with biopsy analysis.
  • Reliable, noninvasive techniques for the diagnosis of EE such as biomarker detection methods, would be preferable to endoscopic techniques.
  • blood levels of potential EE biomarkers such as eosinophils, eotaxin-3, eosinophil-derived neurotoxin, and IL-5 proteins, are known to be elevated in EE, such non-invasive techniques have heretofore not been widely used because their sensitivity and specificity are generally too low to be clinically helpful (see, e.g., Konikoff M. et al. Gastroenterology 131 : 1381-91 (2006)).
  • Eosinophils are multifunctional effector cells produced in the bone marrow from eosinophil lineage-committed progenitor cells. Eosinophils are implicated in the pathogenesis of a variety of diseases, including asthma, hypereosinophilic syndrome, eosinophil gastrointestinal disorders, and parasitic infections, including helminth infection (see, e.g., Broide, D. et al. J. Allergy Clin. Immunol. 127:689-95 (2011); Venge, P. Clin. Respir. J. 4 Suppl. 1 : 15-19 (2010); Anthony, R. et al. Nat. Rev. Immunol. 7:975-87 (2007); Hogan, S. et al. Clin. Exp. Allergy 38:709-50 (2008)).
  • Eosinophils differentiate from hematopoietic stem cells via a common myeloid progenitor cell in mice through an intermediate granulocyte/macrophage progenitor, then via an eosinophil lineage committed progenitor marked by CD34 + and CD125 + (see, e.g., Iwasaki, H. et al. J. Exp. Med. 201 : 1891-7 (2005)).
  • the cytokine IL-5 is particularly important in eosinophil lineage development, as it promotes the selective differentiation of eosinophils and also stimulates the release of mature eosinophils from the bone marrow (see, e.g., Hogan, S.
  • IL-5 has also been shown to promote eosinophil survival by activating MAP kinase, Lyn tyrosine kinase, and PI3 kinase signaling (see, e.g., Kouro, T. et al. Int. Immunol. 21 : 1303-9 (2009); Rosas, M. et al. J. Leukoc. Biol. 80: 186-95 (2006)).
  • EE is associated with marked changes in gene expression, particularly in the esophagus, where -1% of the human genome has an altered, tissue-specific expression pattern, collectively referred to as the EE transcriptome, that is largely but not fully reversible following disease remission with glucocorticoid therapy (see, e.g., Blanchard, C. et al. J. Clin. Invest. 116:536-47 (2006); Blanchard, C. et al. J. Allergy Clin. Immunol. 120: 1292-300 (2007); Abonia, J. et al. J. Allergy Clin. Immunol. 126: 140-9 (2010); Sherrill, J.
  • EE is also an inherited disease that involves a complex combination of genetic and environmental factors (see, e.g., Sherrill, J. and Rothenberg, M. J. Allergy Clin. Immunol. 128:23-32 (2011)).
  • IL- 13 -induced epithelial gene and protein expression changes are central to the pathogenesis of multiple allergic diseases, including EE and asthma.
  • IL-13 is an adaptive immune cytokine that is involved in mediating the effector functions of TR2 responses.
  • the central role of IL-13 in allergic disorders has been demonstrated by the attenuation of experimental allergic diseases in animals with blockade and/or gene deletion of IL-13 and/or its receptor signaling components (see, e.g., Leigh, R. et al. Am. J. Respir. Crit. Care Med. 169:860-7 (2004); Yang, M. et al. J. Immunol. 177:5595-603 (2006); Grunig, G. et al.
  • IL-13-induced gene expression changes in epithelial cells in vitro have been shown to significantly overlap with gene expression changes seen in patients in vivo (see, e.g., Wills-Karp, M. Immunol. Rev. 202:175-90 (2004); Liacouras, C. et al. J. Allergy Clin. Immunol. 128:3-20 (128); Rothenberg, M. Gastroenterology 137: 1238-49 (2009); Blanchard, C. et al. J. Allergy Clin. Immunol. 120: 1292-1300 (2007); Lee, J. et al. Am.
  • the epithelial cell has been shown to be a key target cell type for IL-13 mediated responses, making it an attractive model for investigation. For example, epithelial cells are required for IL-13-induced airway hyper-reactivity and mucus production (Kuperman, D. et al. Nat. Med.
  • miRNAs are associated with EE. Additionally, miRNA expression profiles can be studied in EE, which has a highly conserved, disease- specific transcript profile. A patient's miRNA plasma or serum levels can be measured to provide or contribute to an EE diagnosis; this information can be used to determine an appropriate treatment for the patient.
  • MiRNAs are single-stranded, non-coding RNA molecules of 19-25 nucleotides in length that regulate gene expression post-transcriptionally to silence target genes by either inhibiting protein translation or facilitating the degradation of target mRNAs (see, e.g., Sayed, D. and Abdellatif, M. Physiol. Rev. 91 :827-87 (2011); Winter, J.
  • miRNAs base pair with the complementary regions in the 3' untranslated regions of mRNA and induce translational repression and/or mRNA degradation depending on the degree of complementarity of the base pairing (Carthew, R. and Sontheimer, E. Cell 136:642-55 (2009)).
  • MiRNAs represent a key class of regulators of messenger RNA (mRNA) expression and translation and have diverse roles in fundamental biological processes, such as cell proliferation, differentiation, apoptosis, stress response, and immune response, among many others (see, e.g., Sayed, D. and Abdellatif, M. Physiol. Rev. 91 :827-87 (2011)).
  • MiRNAs represent a particularly attractive class of molecules in the regulation of the EE transcriptome, as a single miRNA can target hundreds of genes and can mediate the epigenetic mechanisms underlying gene-environment interactions, which can have a key but heretofore unexplored role in EE (see, e.g., Sato, F. et al. Febs J. 278: 1598-609 (2011)).
  • miRNA involvement in EE is interesting due to the recent identification of a key role of a specific T helper type 2 (Tn2)-associated miRNA, namely miR-21, in critically regulating T helper cell polarization, as EE involves a local polarized T H 2 response (see, e.g., Blanchard, C.
  • miRNAs 21 upregulated and 11 downregulated miRNAs were identified in patients with active EE, including miR-21 and miR-223 as the most upregulated miRNAs and miR-375 as the most downregulated miRNA in patients with EE. These miRNAs can therefore serve as biomarkers for EE alone or in combination with other biomarkers.
  • miRNAs Three of the differentially regulated miRNAs in the esophageal biopsies, namely miR-146a, miR-146b, and miR-223, were also differentially regulated in EE patient plasma samples; these miRNAs can therefore be used as non-invasive biomarkers for EE alone or in combination with other biomarkers.
  • This EE-associated miRNA signature correlated with the degree of tissue eosinophilia and was distinct from patients with chronic (non-eosinophilic) esophagitis.
  • the differential miRNA expression was largely reversible in patients that responded to glucocorticoid therapy.
  • MiR-21 which has been shown to regulate IL-12 expression and the balance of T H 1 vs. T H 2 responses in mice (see, e.g., Lu, T. et al. J. Immunol. 182:4994-5002 (2009); Lu, T. et al. J. Immunol. 187:3362-73 (2011)), was found to be one of the most up- regulated miRNAs in EE patients. Due to the high level of species conservation of the miR-21 binding site in the 3' untranslated region of IL12p35, miR-21 can have a similar role in human allergic inflammation (see, e.g., Lu, T. et al. J. Immunol. 182:4994-5002 (2009)). The results described herein provide the first set of human data that substantiate that miR-21 can have a similar role in human allergic inflammation.
  • miR-223 and miR-21 were recently found to be up-regulated in eosinophilic esophagitis patients (Lu, T. et al. J. Allergy Clin. Immunol, 129: 1064-1075 el 069 (2012)). They are the top two miRNAs correlated with eosinophil levels in patient esophageal biopsies. Using systems biology analysis, miR-223 and miR-21 were found to co- regulate a set of interacting target genes involved in eosinophil proliferation and differentiation (Lu, T. et al. J. Allergy Clin. Immunol, 129: 1064-1075 el069 (2012)). Because miR-21 promotes cell proliferation, the up-regulation of miR-223 can provide a check and balance in the system given the ability of miR-223 to promote eosinophil maturation.
  • miR-21 * a complementary miRNA of miR-21, was up-regulated after GM-CSF treatment and can inhibit the apoptosis of eosinophils (Wong, C. et al. Immunobiology, 218:255-62 (2013))
  • the minor miRNAs can also have a role in regulating the proliferation of eosinophil progenitors, adding another level of complexity.
  • Therapies targeting miRNAs, including miR-21, miR-223, and their minor miR* forms can allow fine-tuning of the eosinophil level in various diseases.
  • Upregulation of miR-21 in patients with EE can partially explain the increased T H 2 cytokine levels and T H 2 responses seen in EE patients.
  • esophageal miR-21 levels were found to be inversely correlated with esophageal IL-12p35 levels.
  • Up-regulation of miR-21 in EE patients can therefore partially explain the increased T H 2 cytokines and T H 2 responses seen in EE patients.
  • Studies using human data can further elucidate the role of miR-21 in human allergic inflammation.
  • let-7c was found to be down-regulated, meaning let-7c can be used to regulate IL-13 levels (Polikepahad, S. et al. J. Biol. Chem. 285:30139-49 (2010)).
  • Up-regulation of miR-146a was found in EE patients. As miR-146a has recently been demonstrated to selectively regulate Treg-mediated suppression of T H 1 cells (Lu, L. et al. Cell 142:914-29 (2010)), up-regulation of miR-146a can suppress T H 1 responses and promote T H 2 responses.
  • EE One of the defining histological features of EE is intense eosinophil infiltration in the esophagus. As described herein, a majority of the dysregulated miRNAs demonstrate significant correlation between miRNA expression level and esophageal eosinophil count, reflecting disease severity. Functional enrichment analyses were performed for the two miRNAs that most strongly correlated with eosinophil levels, namely miR-21 and miR-223; these analyses empirically predicted that both miRNAs regulate levels of tissue eosinophilia, demonstrating the interplay between these two miRNAs in allergic inflammation.
  • miR-203 is known to repress epithelial cell proliferation and promote epithelial cell differentiation (see, e.g., Yi, R. et al. Nature 452:225-9 (2006)). As such, repression of miR-203 can in part explain the observed epithelial hyperplasia.
  • miRNAs such as miR-21 have been shown to be oncomirs and/or tumor suppressors (see, e.g., Medina, P. et al. Nature 467:86-90 (2010); Hatley, M. et al. Cancer Call 18:282-93 (2010)). While EE is not considered to be a pre -malignant condition, EE involves marked epithelial cell hyperplasia, and these miRNAs can have a role in this feature of EE.
  • miR-675 was found to be the only disease remission- induced miRNA.
  • MiR-675 is derived from the HI 9 gene, which is a paternally imprinted gene (see, e.g., Cai, X. and Cullen, B. RNA 13:313-6 (2007)).
  • the over-expression of HI 9 is commonly associated with various cancers (see, e.g., Tsang, W. Carcinogenesis 31 :350-8 (2010)).
  • HI 9 was previously found to be induced in glucocorticoid responder patients compared to EE patients or normal controls; this induction was not seen in patients that did not respond to glucocorticoid therapy (Caldwell, J. et al. J. Allergy Clin. Immunol. 125:879- 888 (2010)).
  • the miR-675 expression pattern closely resembles that of HI 9. Since the exact roles of HI 9 and its miRNA product, namely miR-675, in the disease remission process have heretofore been unknown, elucidating their functions can provide information regarding the disease remission process in EE. Based on the role HI 9 and miR- 675 can have in DNA methylation responses and the unique overexpression of this miRNA specifically within patients in remission, miR-675 can be involved in epigenetic programming in the esophageal cells of EE remission patients.
  • EE-associated miRNAs were measured.
  • MiR-146a, miR-146b, and miR-223 were found to be up-regulated in the EE plasma samples compared to controls (allergic individuals without EE). These miRNAs can therefore serve as non-invasive biomarkers for EE alone or in combination with other non-invasive biomarkers.
  • Plasma miRNAs have been reported to exist both within exosomes and in protein-bound vesicle-free form (see, e.g., Arroyo, J. et al. Proc. Natl. Acad. Sci. U.S.A. 108:5003-8 (2011); Rabinowits, G. et al. Clin. Lung Cancer 10:42-6 (2009)).
  • the circulating miRNAs can be taken up by cells through exosome uptake or pinocytosis (see, e.g., Valadi, H. et al. Nat. Cell Biol. 9:654-9 (2007); Tian, T. et al. J. Cell. Biochem. 111 :488-96 (2010)).
  • Mast cells have been found to release exosomes containing miRNA (see, e.g., Valadi, H. et al. Nat. Cell Biol. 9:654-9 (2007)). Mast cells also express high levels of both miR-146a and miR-146b (see, e.g., Sonkoly, E. et al. PLoS One 2:e610 (2007); Mayoral, R. et al. J. Immunol. 182:433-45 (2009)). EE patients have concomitant esophageal mastocytosis (see, e.g., Abonia, J. et al. J. Allergy Clin. Immunol. 126: 140-9 (2010); Aceves, S.
  • miR-146a and miR-223 returned to baseline levels during EE remission
  • miR-146b remained elevated in EE remission patients. While the specific role of miR-146b in regulating adaptive immune responses has not been investigated, miR-146a and miR-146b have an identical seed sequence that is critical for miRNA-mediated target gene expression. Therefore, miR-146b can also suppress T R I responses and promote T R 2 responses. EE patients in remission often relapse as time progresses (see, e.g., Assa'ad, A. et al. J. Allergy Clin. Immunol. 119:731-8 (2007)). Therefore, an elevated level of miR-146b can predispose EE patients in remission to a relapse.
  • MiR-21 has been reported to be up-regulated in a variety of disorders associated with eosinophilia, including asthma (see, e.g., Lu, T. et al. J. Immunol. 182:4994-
  • ulcerative colitis see, e.g., Wu, F. et al. Gastroenterology 135: 1624-35 el624
  • MiR-21 has been reported to be pro-pro liferative and anti-apoptotic by targeting multiple tumor suppressor genes (see, e.g., Krichevsky, A. and Gabriely, G. J. Cell. Mol. Med. 13:39-53 (2009); Papagiannakopoulos, T. et al. Cancer Res. 68:8164-72 (2008); Hatley, M. et al. Cancer Cell 18:282-93 (2010)).
  • miR-21 was found to be among the most up-regulated miRNAs in patients with EE and has the highest correlation with esophageal eosinophil levels.
  • MiR-21 has been identified as a regulator of eosinophil progenitor growth and is the first miRNA proven to have a role in directly regulating eosinophil development.
  • MiR-21 was found to be up-regulated during eosinophil differentiation from eosinophil progenitors, and targeted ablation of miR-21 was found to decrease eosinophil progenitor growth.
  • MiR-21 is shown to be progressively up-regulated during IL-5 -driven eosinophil differentiation from progenitor cells in vivo.
  • Eosinophil progenitor cultures derived from miR-2r A mice were found to have increased levels of apoptosis as indicated by increased levels of annexin V positivity compared to those of miR-21 +/+ mice.
  • MiR-21 _/ ⁇ mice were found to have decreased eosinophil colony forming unit capacity in the bone marrow and reduced blood eosinophil levels in vivo. Therefore, targeted ablation of miR-21 in the eosinophil progenitor cultures leads to reduced eosinophil progenitor growth capacity.
  • miR-21 can directly regulate the development of eosinophils by influencing the growth capacity of eosinophil progenitors. Since mature eosinophils lose their proliferative capacity and do not divide, the up-regulation of miR-21 can prevent premature loss of the proliferative potential of eosinophil progenitors. Further elucidation of the roles of miR-21 in regulating eosinophil levels and immunoinflammatory responses can lead to therapeutic options for eosinophilic disorders.
  • miR-21 can exert some effects on direct targets that synergistically interact to ultimately regulate eosinophilopoeisis. Moreover, miR-21 can regulate additional genes at the protein level that were not identified by the genomic screen in the current study. The observed decreased growth capacity of the miR-2r /_ eosinophil progenitors is likely due to modest regulation of a combination of miR-21 targets.
  • Psrcl one of the up-regulated genes, is a predicted target of miR-21 based on sequence conservation and binding site potential (Lu, T. et al. J. Immunol. 182:4994-5002 (2009)). Over-expression of Psrcl has been shown to suppress colony formation in lung carcinoma cells (Lo, P. et al. Oncogene 18:7765-74 (1999)). The up-regulation of Psrcl could potentially contribute to the decreased growth of miR-2r /_ eosinophil progenitors.
  • Pik3r6 a regulatory subunit for phosphoinositide 3 -kinase (PI3 kinase) gamma, was over-expressed in both day 8 and day 12 in the miR-2r /_ eosinophil progenitor cultures.
  • PI3 kinase signaling has been shown to be essential for IL-5 mediated eosinophil survival (Rosas, M. et al. J. Leukoc. Biol. 80: 186-95 (2006)).
  • Pik3r6 has been shown to be expressed primarily in the hematopoietic compartment and can potentially compete with Pik3r5 for binding with ⁇ ⁇ (Suire, S. et al. Curr. Biol.
  • Pik3r6/pl l0y heterodimer is four-fold less sensitive than the Pik3r5/pl l0y heterodimer (Suire, S. et al. Curr. Biol. 15:566-70 (2005)).
  • Pik3r6 and Pik3r5 are expressed at similar levels in the wild type eosinophil progenitors. Up- regulation of Pik3r6 can lead to an increased level of Pik3r6/pl l0y heterodimer and a decreased level of Pik3r5/pl l0y heterodimer, thereby attenuating PI3 kinase signaling. This can in part account for the decreased growth seen in miR-2r /_ eosinophil progenitors.
  • MiR-21 has been known to promote cell growth in various cell types, most notably in tumor cells, by targeting a variety of pro-apoptotic genes both directly and indirectly (Krichevsky, A. and Gabriely, G. J. Cell. Mol. Med. 13:39-53 (2009); Hatley, M. et al. Cancer Cell 18:282-93 (2010)). As described herein, increased levels of apoptosis were found in miR-2r /_ eosinophil cultures compared to miR-21 +/+ cultures. Potential eosinophil hematopoiesis defects were investigated in the miR-2 /_ mice in vivo.
  • the miR-2r /_ mice were found to have both decreased eosinophils in the blood and decreased eosinophil colony forming unit capacity in the bone marrow, consistent with the observed phenotype in the ex vivo eosinophil cultures.
  • MiR-21 has been found to be over-expressed in allergic diseases with significant eosinophilia, including experimental asthma in mice and human EE (Lu, T. et al. J. Immunol. 182:4994-5001 (2009); Wu, F. et al. Gastroenterology 135: 1624-1635 el624
  • MiR-21 was previously found to be capable of regulating immunoinflammatory responses by targeting the IL12/IFNy pathway (Lu, T. et al. J. Immunol. 182:4994-5001
  • miR-21 has been identified as a regulator of eosinophil progenitor growth. This represents the first miRNA demonstrated to have a direct role in regulating eosinophil development. Further elucidating and understanding the roles of miR-21 in regulating the levels of eosinophils and in immunoinflammatory responses can lead to additional therapeutic options for eosinophilic disorders.
  • MiR-223 Deficiency Increases Eosinophil Progenitor Cell Growth
  • MiR-223 has been found to be over-expressed in asthma, EE, and atopic dermatitis, where eosinophils are implicated in the disease pathogenesis to varying degrees (Lu, T. et al. J. Immunol. 182:4994-5002 (2009); Garbacki, N. et al. PLoS One 6:el6509 (2011); Sonkoly, E. et al. J. Allergy Clin. Immunol. 126:581-9 (2010); Mattes, J. et al. Proc. Natl. Acad. Sci. U.S.A. 106: 18704-0 (2009)).
  • MiR-223 has been shown to target the IGFl receptor (IGFIR) (see, e.g., Johnnidis, J. et al. Nature 451 : 1125-9 (2008)), which is the major physiologic receptor for IGFl (see, e.g., Smith, T. Pharmacol. Rev. 62: 199-236 (2010)).
  • IGFl is a major anabolic hormone that stimulates cell growth and is a potent inhibitor of programmed cell death; therefore IGFIR can be differentially regulated by miR-223.
  • IGFl has not been previously examined for its impact on eosinophil progenitors, IGFl and IGFIR inhibitors can be clinically useful for eosinophilic disorders.
  • miR-223 has been shown to be mediated by myeloid transcription factors PU.1 and C/EBP, factors that are important in eosinophilopoiesis (see, e.g., Fukao, T. et al. Cell 129:617-31 (2007)).
  • miR-223 was found to regulate the proliferation and differentiation of eosinophil progenitors; miR-223 was also found to be up-regulated during eosinophil differentiation in an ex vivo bone marrow-derived eosinophil culture model.
  • MiR- 223 -deficient eosinophil progenitor cells were found to have a hyperproliferative capacity.
  • Mechanistic analysis identified a contributory role for the IGFl receptor (IGFIR) in mediating eosinophil progenitor cell proliferation.
  • IGFIR IGFl receptor
  • MiR-223 was found to regulate the growth and differentiation of eosinophil progenitors. MiR-223 was found to be up-regulated in an ex vivo bone marrow-derived eosinophil differentiation culture. Targeted ablation of miR-223 leads to an increase in eosinophil progenitor growth, as miR-223 "7" cells had a markedly increased growth in response to the eosinophil growth factor IL-5. In addition, miR-223 deficiency led to a defect in eosinophil maturation, as indicated by a delayed up-regulation of surface CCR3 expression.
  • IGF 1 R inhibitors are currently under development for the treatment of various types of cancer (Yee, D. Journal of the National Cancer Institute, 104:975-981 (2012)). These data indicate that the IGFIR inhibitors can potentially also be used to treat patients with eosinophilia, such as the hypereosinophilic syndrome (Arefi, M. et al. International Journal of Hematology, 96:320-326 (2012)).
  • miR-223 has been identified as a regulator of eosinophil IGFIR levels. While the up-regulation of IGFIR likely has a contributory role in the increased proliferation seen in the miR-2237 " eosinophil progenitor cultures, this does not preclude the involvement of additional pathways.
  • this microarray analysis identified multiple additional growth and proliferation- related genes differentially regulated between miR-223 + / + and miR-2237 " cultures. These include down-regulation of NAD(P)H:quinone oxidoreductase 1 (NQOl), where NQOl deficient mice have been found to have a significant increase in blood granulocytes including eosinophils (Long, D. et al. Cancer Res., 62:3030-3036 (2002)). Down-regulation of inhibitor of DNA binding 2 (ID2), whose knockdown has been shown to cause increased eosinophil progenitor growth and delayed eosinophil progenitor differentiation, was also observed (Buitenhuis, M. et al. Blood, 105:4272-4281 (2005)).
  • ID2 inhibitor of DNA binding 2
  • IGFIR is expressed by eosinophil progenitors, and an IGFIR inhibitor (pricopodphyllin) inhibits eosinophil progenitor cell growth. Both of these findings occur independent of miR223, although the former is regulated by miR223. Therefore, IGF1 is a new pathway involved in eosinophil development for which pharmacological blockade (independent of miR223) demonstrates a positive effect. This is the first report of the relationship between IGF1 and IGFIR in eosinophilia.
  • NQOl quinone oxidoreductase 1
  • ID2 inhibitor of DNA binding 2
  • NQOl -deficient mice have been shown to have a significant increase in blood granulocytes, including eosinophils (Long, D. et al. Cancer Res. 62:3030-6 (2002)).
  • Silencing of ID2 has been shown to cause increased eosinophil progenitor growth and delayed eosinophil progenitor differentiation (Buitenhuis, M. et al. Blood 105:4272-81 (2005)).
  • miR-223 has been identified as a regulator of eosinophil progenitor proliferation.
  • IGF1R is up-regulated during in eosinophil development
  • miR- 223 is a regulator of IGF1R levels.
  • the roles and regulations of miRNAs during eosinophil development can be utilized to lead to novel therapeutic targets for eosinophilic disorders.
  • MiR-375 Regulates an IL-13 -Induced Epithelial Transcriptome
  • a lentiviral strategy and whole-transcriptome analysis were used in epithelial cells to demonstrate that miR-375 over-expression was sufficient to markedly modify IL-13 -associated immunoinflammatory pathways in epithelial cells in vitro, further substantiating interactions between miR-375 and IL-13.
  • miRNAs have a key role in regulating and fine-tuning IL-13 mediated responses, and miR-375 is a key downstream mediator of IL-13 -induced responses.
  • MiRNA array analysis was used to determine the differentially expressed miRNAs after IL-13 stimulation in two distinct human epithelial cell types, namely esophageal squamous cells and bronchial columnar cells.
  • miR-375 showed a conserved pattern of down-regulation between these two epithelial cell types.
  • MiR-375 levels were analyzed in an IL-13-induced murine asthma model, and down-regulation was observed in the murine asthmatic lungs.
  • miR-375 was inversely related to the degree of allergic inflammation, including esophageal eosinophil levels and gene expression levels of T H 2 cytokines and mast cell specific proteases.
  • MiR-375 over-expression was sufficient to markedly modify IL-13 -associated immunoinflammatory pathways in epithelial cells in vitro.
  • MiRNAs were identified that were differentially regulated after IL-13 stimulation in human bronchial columnar and esophageal squamous epithelial cells.
  • miR-375 was found to be the only miRNA that was down- regulated in both epithelial cell types after IL-13 stimulation in EE patient samples compared to control patients.
  • MiR-375 was found to be inversely correlated with the level of esophageal eosinophils and expression of the mast cell specific genes CP A3 and TPSAB1.
  • the down-regulation of miR- 375 was specific to EE patients; the chronic esophagitis patients had miR-375 expression levels comparable to normal controls.
  • Disease remission with either fluticasone therapy or diet modification was associated with normalization of miR-375 levels; this can be due to the result of reduced IL-13; patients that did not respond to fluticasone therapy continued to have repressed miR-375 levels.
  • miR-375 can potentiate and repress IL-13 -mediated effects, indicating the complex interaction between cytokine and miRNA-mediated gene regulation; miR-375 was therefore sufficient to regulate an IL-13-induced epithelial transcriptome.
  • the inflammatory diseases and immunological diseases are the two most significantly over- represented disease states regulated by miR-375. These include allergy-associated genes, such as MMP12 and MUC4 (Mukhopadhyay, S. et al. J. Allergy Clin. Immunol. 126:70-6 (2010); Lavigne, M. et al. Biochem. Biophys. Res. Comm.
  • miR-375 expression was found to be unchanged after 2 hours of IL-13 stimulation, despite the finding of up-regulation of miR-375 after 2 hours of IL-13 stimulation reported in a previous study (Biton, M. et al. Nat. Immunol. 12:239-46 (2011)). This inconsistency could be due to the use of the HT-29 human colon adenocarcinoma cell line, as opposed to human esophageal squamous cells and bronchial columnar cells.
  • MiR-375 was found to be down-regulated after 24 and 48 hours of IL-13 stimulation, which is consistent with the previous finding that miR-375 levels were at or below baseline after 16 hours of IL-13 stimulation (Biton, M. et al. Nat. Immunol. 12:239-46 (2011)).
  • miR-375 down-regulation has been reported in patient samples from multiple T H 2-associated diseases, such as atopic dermatitis (Sonkoly, E. et al. J. Allergy Clin. Immunol. 126:581-9 (2010)) and ulcerative colitis (Wu, F. et al. Gastroenterology 135: 1624- 35 (2008)), and hyperproliferative diseases, such as esophageal squamous carcinoma (Kong, K. et al. Gut 61 :33-42 (2011)), up-regulation of miR-375 in a T H 2-associated disease in humans has not been reported. Therefore, IL-13 can have the long-term effect of down- regulating miR-375 expression.
  • MiR-375 has been previously shown to enhance goblet cell differentiation by repressing KLF5 expression (Biton, M. et al. Nat. Immunol. 12:239-46 (2011)) and has also been shown to attenuate cell proliferation by targeting IGF1R, PDK1, and YWHAQ (Kong, K. et al. Gut 70:2239-49 (2011); Tsukamoto, Y. et al. Cancer Res. 70:2339-49 (2010)). As described herein, neither of these pathways was affected in EE patients or upon analysis of miR-375 -regulated genes in esophageal epithelial cells (Blanchard, C. et al. J. Clin. Invest. 116:536-47 (2006)), indicating that the activity of miR-375 may be dependent on the cellular context, consistent with previous reports (Tsukamoto, Y. et al. Cancer Res. 70:2339-49
  • MiR-375 has been previously reported to regulate TSLP expression in an HT-29 human colonic adenocarcinoma cell line (Biton, M. et al. Nat. Immunol. 12:239-46
  • TSLP and miR-375 were concomitantly induced by IL-13 in HT-29 cells, and knockdown of miR-375 inhibited TSLP production.
  • over-expression of miR-375 induced TSLP expression in HT-29 cells (Biton, M. et al. Nat. Immunol. 12:239-46 (2011)).
  • TSLP has been shown to have an important role in EE pathogenesis (see, e.g., Rothenberg, M. et al. Nat. Genet. 42:289-91 (2010); Sherrill, J. et al. J. Allergy Clin. Immunol. 126: 160-5 (2010)).
  • miR-375 As described herein, the ability of miR-375 to regulate TSLP expression in esophageal epithelial cells was analyzed. Additionally, miR-375 was found to have no effect on TSLP production, and there was no correlation between miR-375 and TSLP in the esophageal samples. The disparity between these results and previous studies could be due to the use of different cell types and/or different mechanisms in TSLP induction in these cells, since previous studies report that IL-13 induces TSLP expression in HT-29 cells but not in esophageal epithelial cells (Blanchard, C. et al. J. Allergy Clin. Immunol. 120: 1292-1300 (2007); Biton, M. et al. Nat. Immunol. 12:239-46 (2011)).
  • IL-13 has been found to down-regulate miR-375 and modulate IL-13 regulated gene expression in bronchial epithelial cells; this is relevant to asthma and other IL- 13-mediated diseases.
  • miR-375 it is unclear whether over-expression of miR-375 can correct the allergic phenotype in asthma and EE. This can be resolved in future studies utilizing miR- 375 lung and/or esophageal epithelial specific transgenic mice.
  • MiR-375 expression levels were found to reflect disease activity, normalize with remission, and inversely correlate with the degree of allergic inflammation.
  • MiR-375 was strongly associated with parameters germane to allergic responses, including eosinophil levels, gene expression levels of the T H 2 cytokines IL-5 and IL-13, the mast cell-specific enzymes CPA3 and TPSAB1, and POSTN (the gene that encodes periostin).
  • Periostin has been demonstrated to have a key role in IL-13 associated remodeling responses (Blanchard, C. et al. Mucosal. Immunol. 1 :289-96 (2008)), and its level predicts responsiveness to anti-IL-13 therapy in humans (Corren, J. et al. N.
  • Embodiments of the invention are directed to methods of treating EE in a patient, wherein the methods comprise analyzing the sample from a patient to determine a level of one or more miRNAs associated with EE, determining whether the level of the one or more miRNAs is up-regulated or down-regulated relative to a level of the one or more miRNAs measured in a normal individual, wherein the presence of an elevated or reduced level of one or more miRNAs associated with EE results in the patient being diagnosed with EE, and treating the patient with an appropriate therapeutic strategy based upon the diagnosis.
  • Embodiments of the invention are also directed to methods of distinguishing EE from other disorders in a subject, wherein the methods comprise analyzing the sample from a patient to determine a level of one or more miRNAs associated with EE, determining whether the level of the one or more miRNAs is up-regulated or down-regulated relative to a level of the one or more miRNAs measured in a normal individual, wherein the presence of an elevated or reduced level of one or more miRNAs associated with EE results in the patient being diagnosed with EE or with another disorder, and treating the patient with an appropriate therapeutic strategy based upon the diagnosis.
  • the other disorder is chronic esophagitis.
  • Embodiments of the invention are also directed to methods of determining whether a subject with EE has active EE or remission EE, wherein the methods comprise analyzing the sample from a patient to determine a level of one or more miRNAs associated with EE, determining whether the level of the one or more miRNAs is up-regulated or down- regulated relative to a level of the one or more miRNAs measured in a normal individual, wherein the presence of an elevated or reduced level of one or more miRNAs associated with EE results in the patient being diagnosed with active EE or remission EE, and treating the patient with an appropriate therapeutic strategy based upon the diagnosis.
  • the one or more EE-associated miRNAs are selected from the list of genes dysregulated in EE provided in Figure 1.
  • At least one miRNA is selected from Figure 1. In some embodiments the at least one miRNA includes miR-21. In some embodiments, at least one miRNA is selected from Figure 1. In some embodiments the at least one miRNA includes miR-21. In some embodiments the at least one miRNA includes miR-223. In some embodiments the at least one miRNA includes miR-375. In some embodiments the at least one miRNA includes miR-146a. In some embodiments the at least one miRNA includes miR- 146b.
  • At least 2 miRNAs are selected from Figure 1. In some embodiments, at least 3 miRNAs are selected from Figure 1. In some embodiments, at least 4 miRNAs are selected from Figure 1. In some embodiments, at least 5 miRNAs are selected from Figure 1. In some embodiments, at least 10 miRNAs are selected from Figure 1. In some embodiments, at least 15 miRNAs are selected from Figure 1. In some embodiments, at least 20 miRNAs are selected from Figure 1. In some embodiments, at least 25 miRNAs are selected from Figure 1. In some embodiments, at least 30 miRNAs are selected from Figure 1. In some embodiments, all of the miRNAs are selected from Figure 1.
  • 1, 2, 3, 4, 5, 6, 7, 8, or 9 miRNAs are selected from Figure 1.
  • 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 miRNAs are selected from Figure 1.
  • 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 miRNAs are selected from Figure 1.
  • 30, 31, or 32 miRNAs are selected from Figure 1.
  • any between 1 to 10 miRNAs are selected from Figure 1. In some embodiments, anywhere between 1-20 miRNAs are selected from Figure 1. In some embodiments, anywhere between 1-30 miRNAs are selected from Figure 1.
  • the miRNAs associated with EE are measured using one or more methods and/or tools, including for example, but not limited to, Taqman (Life Technologies, Carlsbad, CA), Light-Cycler (Roche Applied Science, Penzberg, Germany), ABI fiuidic card (Life Technologies), NanoString® (NanoString Technologies, Seattle, WA), NANODROP® technology (Thermo Fisher Scientific (Wilmington, DE), fiuidic card, and the like.
  • Taqman Life Technologies, Carlsbad, CA
  • Light-Cycler Roche Applied Science, Penzberg, Germany
  • ABI fiuidic card Life Technologies
  • NanoString® NanoString Technologies, Seattle, WA
  • NANODROP® technology Thermo Fisher Scientific (Wilmington, DE), fiuidic card, and the like.
  • the person of skill in the art will recognize such other formats and tools, which can be commercially available or which can be developed specifically for such analysis.
  • Determination of the miRNA level(s) as described herein can be combined with determination of the levels of one or more non-miRNA biomarkers associated with EE.
  • determination of the miRNA level(s) as described herein can be combined with determination of the levels of one or more genes of the EE transcriptome.
  • Such a determination can include measurement of the gene DNA or RNA, or the gene product.
  • genes can include, for example, eotaxin-3, and the like.
  • Embodiments of the invention are also directed to methods of treating an eosinophilic disorder (other than EE) in a patient, wherein the methods comprise analyzing the sample from a patient to determine a level of one or more miRNAs associated with an eosinophilic disorder, determining whether the level of the one or more miRNAs is up- regulated or down-regulated relative to a level of the one or more miRNAs measured in a normal individual, wherein the presence of an elevated or reduced level of one or more miRNAs associated with an eosinophilic disorder results in the patient being diagnosed with an eosinophilic disorder, and treating the patient with an appropriate therapeutic strategy based upon the diagnosis.
  • Eosinophilic disorders other than EE include, for example, eosinophilic gastrointestinal disorder (EGID) outside of the esophagus, asthma, and the like.
  • miRNAs associated with asthma include miR375.
  • the method of Claim 29, wherein the one or more miRNAs associated with asthma comprises miR375.
  • the sample comprises lung and/or lung epithelial cells.
  • EE therapies include the use of proton pump inhibitors (PPIs), topical glucocorticoids, such as fluticasone, budesonide, or ciclesonide, humanized antibodies against relevant cytokines and/or mediators, such as eotaxin-1, eotaxin- 3, IL-13, IL-5, IL-5Ra, CD49D, SIGLEC-8, IgE, CD300A, TSLP, and/or IL-33, small molecule inhibitors of an eosinophil and/or allergic disease activation pathway, such as a notch-signaling inhibitor or an inhibitor or antagonist of CCR3, CCL11, VLA4, CRT R 2, prostaglandin D2, histamine H4 receptor, IL-13, IL-4, and/or the common ⁇ chain, and small molecule inhibitors
  • PPIs proton pump inhibitors
  • topical glucocorticoids such as fluticasone, budesonide, or ciclesonide
  • EE can be treated through the blockade of eosinophil recruitment, such as through CCR3 and/or CCL11 inhibition, adhesion molecule inhibition, CRTH2 and prostaglandin D2 inhibition, histamine H4 receptor inhibition, IL-13 and/or IL-4 blockade, and the like.
  • Compounds that can be used for these purposes include, for example, small molecule CCR3 antagonists and/or eotaxin-1 -specific antibodies for CCR3 and/or CCL11 inhibition, CD49D-specific antibodies and/or small molecule VLA4 antagonists for adhesion molecule inhibition, CRT R 2 antagonists for CRT R 2 and prostaglandin D2 inhibition, small molecule histamine H4 receptor antagonists for histamine H4 receptor inhibition, and IL-13 -specific antibodies, IL-4Ra antagonists, IL-4 variants for IL-13 and/or IL-4 blockade, and the like.
  • Such compounds include, for example, small molecule CCR3 antagonists, such as LH31407, eotaxin-1 -specific antibodies, such as bertilimumab, CD49D-specific antibodies, such as natalizubam, small molecule VLA4 antagonists, such as compound 1, CRT H 2 antagonists, such as OC000459, small molecule histamine H4 receptor antagonists, such as INCB38579, IL-13 -specific antibodies, such as lebrikizumab, IL-4Ra antagonists, such as AMG 317, IL-4 variants, such as pitrakinra, and the like.
  • small molecule CCR3 antagonists such as LH31407
  • eotaxin-1 -specific antibodies such as bertilimumab
  • CD49D-specific antibodies such as natalizubam
  • small molecule VLA4 antagonists such as compound 1
  • CRT H 2 antagonists such as OC000459
  • EE can be treated through the inhibition of eosinophil survival, such as through IL-5 and/or IL-5Ra blockade, SIGLEC-8 agonism, IgE blockade, activation of inhibitory receptors, TSLP inhibition, and the like.
  • Compounds that can be used for these purposes include, for example, IL-5-specific antibodies, IL-5Ra-specific antibodies, and/or antisense oligonucleotides directed against the common ⁇ chain for IL-5 and/or IL-5Ra blockade, SIGLEC-8-specific antibodies for SIGLEC-8 agonism, IgE-specific antibodies for IgE blockade, CD300A-specific antibodies for activation of inhibitory receptors, TSLP-specific antibodies for TSLP inhibition, and the like.
  • IL-5-specific antibodies such as mepolizumab and reslizumab
  • IL-5Ra-specific antibodies such as benralizumab
  • antisense oligonucleotides directed against the common ⁇ chain such as TPI ASM8, SIGLEC-8-specific antibodies
  • IgE- specific antibodies such as omalizumab
  • CD300 A- specific antibodies such as CD300 A- specific antibodies
  • TSLP-specific antibodies such as AMG 157, and the like
  • EE can be treated through the inhibition of eosinophil activation, such as through IL-33 blockade, notch inhibition, and the like.
  • Compounds that can be used for these purposes include, for example, IL-33 -specific antibodies for IL-33 blockade, notch signaling inhibitors for notch inhibition, and the like.
  • Specific examples of such compounds include, for example, IL-33 -specific antibodies, notch signaling inhibitors, such as semagacestat, and the like
  • EE can be treated through the blockade of eosinophil production, such as through IL-5R blockade, and the like.
  • Compounds that can be used for these purposes include, for example, IL-5Ra-specific antibodies for IL-5R blockade, and the like. Specific examples of such compounds include, for example, IL-5Ra-specific antibodies, such as benralizumab, and the like.
  • miRNAs or modified miRNAs as therapeutic targets or agents.
  • any miRNA(s) associated with EE found to be elevated relative to the level(s) of the one or more miRNAs measured in a normal individual or using one or more corresponding modified miRNA(s) can be used as a therapeutic target or agent.
  • EE can be treated by modulating one or more miRNAs via one or more of a number of approaches, including the use of anti-miRNA oligonucleotides (antagomirs, or AMOs), antisense oligonucleotides (ASOs), locked nucleic acids (LNAs) which modify miRNAs or serve as modified antisense oligonucleotides, RNA competitive inhibitors or decoys (miRNA sponges), small molecule inhibitors of miRNA stem-loop processing, and viral vectors expressing one or more miRNA genes, including lentiviral vectors (LVs), adenoviral vectors (AVs), and adeno-associated virus (AAV), and the like.
  • AMOs anti-miRNA oligonucleotides
  • ASOs antisense oligonucleotides
  • LNAs locked nucleic acids
  • miRNA sponges small molecule inhibitors of miRNA stem-loop processing
  • EE therapy can involve the administration of an antagomir directed against a miRNA found to be elevated relative to the level(s) of the one or more miRNAs measured in a normal individual, a miR-21, miR-223, miR-146a, and/or miR-146b antagomir, an IGFl or IGF1R inhibitor, such as NVP-AEW541 and/or pricopodophyllin, and the like.
  • anti-miRNA therapeutics can be developed by the screening of various compounds.
  • Compounds that can be screened to determine their utility as anti-miRNA therapeutics include for example, but are not limited to, libraries of known compounds, including natural products, such as plant or animal extracts, synthetic chemicals, biologically active materials including proteins, peptides such as soluble peptides, including but not limited to members of random peptide libraries and combinatorial chemistry derived molecular libraries made of D- or L-configuration amino acids, or both, phosphopeptides (including, but not limited to, members of random or partially degenerate, directed phosphopeptide libraries), antibodies (including, but not limited to, polyclonal, monoclonal, chimeric, human, anti-idiotypic or single chain antibodies, and Fab, F(ab') 2 and Fab expression library fragments, and epitope-binding fragments thereof), organic and inorganic molecules, and the like.
  • a model can also be generated by building models of the hydrophobic helices. Mutational data that point towards residue-residue contacts can also be used to position the helices relative to each other so that these contacts are achieved. During this process, docking of the known ligands into the binding site cavity within the helices can also be used to help position the helices by developing interactions that would stabilize the binding of the ligand.
  • the model can be completed by refinement using molecular mechanics and loop building using standard homology modeling techniques. (General information regarding modeling can be found in Schoneberg, T. et. al. Molecular and Cellular Endocrinology 151 :181-93 (1999); Flower, D. Biochimica et Biophysica Acta 1422:207-34 (1999); and Sexton, P. Current Opinion in Drug Discovery and Development 2:440-8 (1999).)
  • the model can be used in conjunction with one of several existing computer programs to narrow the number of compounds to be screened by the screening methods of the present invention, like the DOCK program (UCSF Molecular Design Institute, San Francisco, CA). In several of its variants it can screen databases of commercial and/or proprietary compounds for steric fit and rough electrostatic complementarity to the binding site.
  • Another program that can be used is FLEXX (Tripos Inc., St. Louis, MO).
  • Any anti-gastroesophgeal reflux disease (GERD) therapy can be used to treat chronic esophagitis.
  • GERD gastroesophgeal reflux disease
  • esophagitis histological finding of epithelial hyperplasia with acute non-eosinophilic inflammation
  • others are purely clinical (heartburn)
  • others are based on measurement of esophageal acid levels (e.g. pH probes).
  • GERD and chronic (non-eosinophilic) esophagitis can be considered to be equivalent as a first approximation, and anti-GERD therapies can be used to treat chronic esophagitis.
  • Anti-GERD therapies include, for example, antacid administration, H2 agonist administration, and/or PPI therapy, and the like. Certain embodiments of the invention involve administering chronic esophagitis therapies, including antacid administration, H2 agonist administration, and/or PPI therapy.
  • the miRNAs, modified miRNAs, or anti-miRNAs used as therapeutic targets or agents can be administered via oral or parenteral delivery routes (subcutaneous or intravenous), as has been described previously (van Rooij, E. et al. Circ. Res. 110:496-507 (2012)).
  • Such therapeutics can be administered by any pharmaceutically acceptable carrier, including, for example, any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • any pharmaceutically acceptable carrier including, for example, any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional medium or agent is incompatible with the active compound, such media can be used in the compositions of the invention.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • Routes of administration include for example, but are not limited to, intravenous, intramuscular, and oral, and the like. Additional routes of administration include, for example, sublingual, buccal, parenteral (including, for example, subcutaneous, intramuscular, intraarterial, intradermal, intraperitoneal, intracisternal, intravesical, intrathecal, or intravenous), transdermal, oral, transmucosal, and rectal administration, and the like.
  • Solutions or suspensions used for appropriate routes of administration can include, for example, the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediammetetraacetic acid; buffers such as acetates, citrates, or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose, and the like.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
  • antibacterial agents such as benzyl alcohol or methyl parabens
  • antioxidants such as ascorbic acid or sodium bisulfate
  • chelating agents such as
  • the pH can be adjusted with acids or bases, such as, for example, hydrochloric acid or sodium hydroxide, and the like.
  • the parenteral preparation can be enclosed in, for example, ampules, disposable syringes, or multiple dose vials made of glass or plastic, and the like.
  • compositions suitable for injectable use include, for example, sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion, and the like.
  • suitable carriers include, for example, physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS), and the like.
  • the composition should be fluid to the extent that easy syringability exists.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof, and the like.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents such as, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride, and the like, in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption such as, for example, aluminum monostearate and gelatin, and the like.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets, for example.
  • the agent can be contained in enteric forms to survive the stomach or further coated or mixed to be released in a particular region of the gastrointestinal (GI) tract by known methods.
  • the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, or the like.
  • Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.
  • Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches, and the like can contain any of the following exemplary ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel®, or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring, or the like.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel®, or corn starch
  • a lubricant such as magnesium stearate
  • a glidant such as colloidal silicon dioxide
  • the compounds can be delivered in the form of an aerosol spray from pressured container or dispenser, which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer, or the like.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer, or the like.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives, and the like.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems, and the like.
  • a controlled release formulation including implants and microencapsulated delivery systems, and the like.
  • Biodegradable, biocompatible polymers can be used, such as, for example, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, and the like. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,81 1 , which is incorporated herein by reference in its entirety.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the details for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals. Such details are known to those of skill in the art.
  • Certain embodiments of the invention include using quantification data from a gene-expression analysis and/or from a miRNA analysis, either from an esophageal biopsy sample, or from a sample of esophageal mucosa, or from a blood sample.
  • Embodiments of the invention include not only methods of conducting and interpreting such tests but also include reagents, kits, assays, and the like, for conducting the tests.
  • Diagnostic-testing procedure performance is commonly described by evaluating control groups to obtain four critical test characteristics, namely positive predictive value (PPV), negative predictive value (NPV), sensitivity, and specificity, which provide information regarding the effectiveness of the test.
  • the PPV of a particular diagnostic test represents the proportion of subjects with a positive test result who are correctly diagnosed; for tests with a high PPV, a positive test indicates the presence of the condition in question.
  • the NPV of a particular diagnostic test represents the proportion of subjects with a negative test result who are correctly diagnosed; for tests with a high NPV, a negative test indicates the absence of the condition.
  • Sensitivity represents the proportion of correctly identified subjects who are actual positives; for tests with high sensitivity, a positive test indicates the presence of the condition in question. Specificity represents the proportion of correctly identified subjects who are actual negatives; for tests with high specificity, a negative test indicates the absence of the condition.
  • the correlations disclosed herein, between EE and miRNA levels and/or mRNA levels and/or gene expression levels, provide a basis for conducting a diagnosis of EE, or for enhancing the reliability of a diagnosis of EE by combining the results of a quantification of miRNA with results from other tests or indicia of EE.
  • the results of a quantification of miRNA could be combined with the results of a quantification of one or more cytokines or mRNAs.
  • the correlation can be one indicium, combinable with one or more others that, in combination, provide an enhanced clarity and certainty of diagnosis. Accordingly, the methods and materials of the invention are expressly contemplated to be used both alone and in combination with other tests and indicia, whether quantitative or qualitative in nature.
  • p values below 0.05 are considered to be statistically significant, it is within the scope of embodiments of the present invention to make use of correlations having a reported p value above 0.05 as well as below 0.05.
  • a p value can be above 0.05, such as, for example, 0.06, 0.07, 0.08, 0.09, 0.10, 0.15, or more.
  • p value is affected by sample size, two studies can have the same proportion of outcomes, and a study with a smaller sample size can have a p value above 0.05, while the study with the larger sample size can have a p value below 0.05, even though the correlation is proportionally the same.
  • a p value below 0.05 for any sample size, is a strong indication of a statistically significant correlation, a genuine correlation can exist, that is tested with a small sample size, and the p value of such a test can be above 0.05.
  • the inclusion criteria for active chronic esophagitis patients include a clinical diagnosis of esophagitis and eosinophil counts of 1-15 per 400x hpf in the esophageal biopsies. Patients with systemic or swallowed topical glucocorticoid use were excluded from the selection of active EE or active chronic esophagitis patients.
  • the inclusion criteria for EE patients responding to glucocorticoid treatment included a history of EE, treatment with swallowed topical glucocorticoid, response as indicated by eosinophil count ⁇ 2 per 400x hpf, and normalization of histological features of the disease.
  • Table 1 Patient clinical characteristics.
  • RNA, including miRNA, from patient esophageal biopsy samples was isolated using the miRNeasy Mini Kit (Qiagen, Valencia, CA), according to the manufacturer's instructions. RNA quality was assessed using the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA), and only samples with RNA integrity number > 8 were included in the analyses. MiRNA expression was profiled using the TaqMan Human MicroRNA Array v2.0 (Applied Biosystems, Carlsbad, CA), which includes probes for 667 human miRNAs, as annotated in release 10.0 of the miRBase microRNA Registry (Griffiths- Jones, S. et al. Nucleic Acids Res.
  • microarray data have been deposited into the Array Express database, found at http ⁇ colon slash slash> www ⁇ dot> ebi ⁇ dot> ac ⁇ dot> uk ⁇ slash> arrayexpress, with accession number E-MEXP-3298, in compliance with minimum information about microarray experiment (MIAME) standards.
  • MiRNAs differentially regulated between healthy control and EE patients were identified by normalizing the expression data to the average of two endogenous control probes, namely U6 and RNU44, then filtered on cycle threshold values ⁇ 30 and at least a 2- fold change between normal controls and EE patients. Statistical significance was determined at P ⁇ .05 with Benjamini Hochberg false discovery rate correction. The list of differentially expressed miRNAs was clustered using hierarchical clustering, and a heatmap was generated. Similar analyses were carried out comparing normal controls to chronic esophagitis patients, and patients that did not received glucocorticoid treatment to patients that received glucocorticoid treatment to identify differentially expressed miRNAs between the groups. qRT-PCR for miRNA
  • Esophageal biopsy specimens from patients with EoE and healthy control subjects were profiled with the TaqMan Human miRNA Array V2.0, comprising 677 miRNAs, as annotated in version 10 of the miRBase registry, to identify miRNAs differentially expressed in patients with EoE (Griffiths- Jones, S. et al. Nucleic Acids Res. 36:D154-8 (2008)).
  • 677 miRNAs assayed 254 miRNAs were expressed above background levels (Table 2).
  • a comparison between normal controls and EE patients identified 21 up-regulated and 11 down-regulated miRNAs ( Figure 1).
  • the most up-regulated miRNAs included miR-21 and miR-223, and the most down-regulated miRNA was miR-375.
  • the differentially expressed miRNAs were evaluated by performing quantitative real time polymerase chain reaction (qRT-PCR) on a selected set of differentially expressed miRNAs, including miR-21, miR-223, miR-375, let-7c, and miR-203 ( Figure 2A- E). There was a strong correlation between the qRT-PCR and microarray data, with a Pearson correlation coefficient of 0.99 and p ⁇ 0.01 ( Figure 2F).
  • qRT-PCR quantitative real time polymerase chain reaction
  • hsa-miR-143 0.420 -0.177 -0.180 -0.706 hsa-miR-144* -0.1 12 -0.996 -1.429 0.021 hsa-miR-145* 0.377 -0.590 -0.71 1 -0.982 hsa-miR-145 0.184 -0.904 -1.032 -1.493 hsa-miR-146a 0.150 0.114 1.635 -0.280 hsa-miR-146b 0.042 0.249 2.153 -0.046 hsa-miR-148a -0.059 0.132 -0.365 0.372 hsa-miR-148b* 0.072 -0.064 0.174 0.140 hsa-miR-148b 0.024 0.067 -0.033 0.292 hsa-miR-149 0.021 0.007 -0.366 -0.230 hsa-miR-150 -0.1
  • Example 1 A subsequent study was undertaken to determine whether the miRNA expression signature identified in Example 1 was specific to EE.
  • the miRNA expression profile of EE patients was compared to that of healthy controls, as well as patients who presented with symptoms of EE but were ultimately diagnosed with chronic (non- eosinophilic) esophagitis.
  • the chronic esophagitis patients had a miRNA expression profile that was similar to normal healthy controls and distinct from EE patients ( Figure 3 A). No miRNAs that were differentially regulated between normal controls and chronic esophagitis patients were identified.
  • Esophageal mRNA from EE patients was reverse-transcribed to cDNA by using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) following the manufacturer's protocol, using the TaqManreagents for amplification of EE signature genes (Applied Biosystems) (Abonia, J. et al. J. Allergy Clin. Immunol. 126: 140-9 (2010); Blanchard, C. et al. J. Clin. Invest. 116:536-47 (2006); Blanchard, C. et al. J. Allergy Clin. Immunol. 120: 1292-300 (2007)).
  • the EE signature genes are listed in Table 4.
  • Table 4 List of EE signature genes used for correlation analysis.
  • MiR-21 was found to be significantly correlated with the esophageal expression of genes involved in inflammation, including CCL26 (eotaxin-3), remodeling including POSTN (periostin), eosinophilia including IL-5, and cell specific markers for eosinophils (CLQ and mast cells ⁇ CP A3 and TPSAB1) ( Figure 3B).
  • miR-21 significantly correlated with the gene CTNNAL1, which has been implicated in cell growth, proliferation, and wound repair (Figure 3B) (Xiang, Y. et al. J. Cell. Biochem. 103:920-30 (2008)).
  • MiR-223 had the highest correlation with POSTN, IL-5, and CLC ( Figure 3C).
  • MIR-675 IS FOUND TO BE A DISEASE REMISSION-INDUCED MIRNA IN EE
  • miR-675 is a glucocorticoid-induced or EE remission-induced miRNA
  • miR-675 expression levels were measured in normal controls, EE patients, EE patients that responded to glucocorticoid treatment, and EE patients that did not respond to glucocorticoid treatment, as described in Example 1.
  • MiR-675 was not induced in patients that did not respond to glucocorticoid treatment (Figure 4B).
  • miR-21 and miR-223 were found to be the top two upregulated miRNAs in patients with EE, a subsequent study was undertaken to identify mRNA expression patterns that significantly correlated with the expression of miR-21 and miR-223. The study was conducted on esophageal RNA samples subjected to RNA-Seq analysis, as well as previously published mRNA gene expression microarray experiments (see, e.g., Blanchard, C. et al. J. Clin. Invest. 1 16:536-47 (2006); Abonia, J. et al. J. Allergy Clin. Immunol. 126: 140-9 (2010)).
  • RNA-Seq data were obtained from esophageal RNA samples from 11 EE patients by the Genomic Sequencing Core Laboratory at Cincinnati Children's Hospital Medical Center.
  • the RNA-Seq was aligned to the GrCh37 build of the human genome using the Ensembl annotations as a guide for TopHat (Johns Hopkins University, Baltimore, MD; University of California, Berkeley, CA; Harvard University, Cambridge, MA) (Trapnell, C. et al. Bioinformatics 25: 1105-11 (2009)).
  • the resulting files were then analyzed with Cufflinks ((Johns Hopkins University, Baltimore, MD; University of California, Berkeley, CA; California Institute of Technology) to test for differential expression and differential regulation (Trapnell, C. et al. Nat. Biotechnol. 28:511-1 (2010)).
  • the 3' region of miR-21 was identified as the best representation of pri- miR-21 (labeled as exon 3 in Figure 5 A).
  • the region 3' of miR-223 was identified as the best representation of pri-miR-223 (labeled as exon 2 in Figure 5B) (Saini, H. et al. BMC Genomics 9:564 (2008)).
  • the pri-miR-21 region was found to be significantly enriched in the EE patients compared to the control patients.
  • the expression pattern of the pri-miR-21 was then correlated with the other gene expression patterns present from the RNA-Seq expression profiles.
  • MiR-223 was analyzed in a similar fashion.
  • Gene expression microarray experiments from EE patients were also analyzed (Abonia, J. et al. J. Allergy Clin. Immunol. 126: 140-9 (2010); Blanchard, C. et al. J. Clin. Invest. 116:536-47 (2006)).
  • MiR-21 and miR-223 were correlated using the Human U133 probes (Affymetrix, Santa Clara, CA) that best corresponded to the miR A.
  • a correlated gene is one whose expression follows similar or inverse trends with the miRNA in question.
  • These probes were then correlated with microarray expression data, and 470 probes correlated well with at least one of the miRNA probes.
  • the genes that showed the highest correlations and the most highly differentially regulated genes in the previously published EE transcriptome were used as a training set alongside genes related to T H 1/T R 2 differentiation and eosinophilia that are target genes of miR-21 and miR-223 (Chen, J. et al. Nucleic Acids Res.
  • MiR-21 and miR-223 were found to interactively regulate many similar pathways, including leukocyte proliferation, leukocyte activation, cytokine production, and immune response (Figure 6A).
  • the co-regulated target genes of miR-21 and miR-223 were involved in adaptive immune system polarization, IFNy signaling, and regulation of eosinophilia ( Figure 6B, Figure 7).
  • the significantly enriched pathways included regulation of interleukin secretion, interferon production and signaling, and T-cell differentiation and activation ( Figure 6B).
  • Esophageal IL-12p35 levels showed a strong inverse correlation with esophageal miR-21 levels ( Figure 6C).
  • MiRNAs have recently been reported to be present in plasma samples in a stable form protected from endogenous RNAse activities (Mitchell, P. et al. Proc. Natl. Acad. Sci. U.S.A., 105:10513-8 (2008)). Plasma miRNAs can therefore be used as noninvasive biomarkers ((Mitchell, P. et al. Proc. Natl. Acad. Sci. U.S.A., 105: 10513-8 (2008); Zahm, A. et al. J. Pediatr. Gastroenterol. Nutr., 53:26-33 (2011); Shen, J. et al. Lab. Invest., 91 :579-87 (2011)). Accordingly, a study was undertaken to evaluate a subset of the miRNAs found to be differentially regulated in the esophageal biopsies of EE patients to determine whether these miRNAs could also be differentially regulated in patient plasma samples.
  • normal atopic controls defined as patients having asthma, allergic rhinitis, and/or eczema
  • the active EE patients were selected based on a clinical diagnosis of EE and eosinophil counts of > 24 per 400x hpf in the esophageal biopsies. Patients with systemic or swallowed topical glucocorticoid use were excluded from the selection of normal atopic control or active EE patients.
  • the EE remission patients were selected based on a history of EE and treated with swallowed topical glucocorticoid, with response as indicated by eosinophil count ⁇ 1 per 400x hpf and normalization of histological features of the disease.
  • RNA from the bacteriophage MS2 was selected as a carrier RNA because it does not contain miRNAs.
  • the RNA was then extracted using the miRNEasy Mini Kit (Qiagen) according to the manufacturer's protocols.
  • Plasma prepared from Na-EDTA tubes
  • miRNAs were reverse transcribed using MegaPlex RT primers (Applied Biosystems), Human pool set V2.1 with pre- amplification (Applied Biosystems), according to the manufacturer's protocols.
  • the pre- amp lifted samples were diluted 1 :40, and 1.5 ⁇ of the diluted product was then used in a 15 ⁇ PCR reaction using the TaqMan miRNA Assays (Applied Biosystems), following the manufacturer's protocols.
  • 00220 Using plasma samples from EE patients and normal controls, the expression levels of the 10 most differentially regulated miRNAs associated with EE were determined. This analysis included 6 up-regulated miRNAs, namely miR-21, miR-132, miR-142-3p, miR- 146a, miR-146b, and miR-223, and four down-regulated miRNAs, namely miR-203, miR- 210, miR-365, and miR-375.
  • up-regulated miRNAs namely miR-21, miR-132, miR-142-3p, miR- 146a, miR-146b, and miR-223, and four down-regulated miRNAs, namely miR-203, miR- 210, miR-365, and miR-375.
  • miR-16 has been reported to have a constant expression level in plasma samples (Shen, J. et al. Lab. Invest. 91 :579-87 (2011)), miR-16 was used as an endogenous control to determine the differential expression of the miRNAs between EE patient plasma samples and normal controls. There were no significant differences in the average C T value of miR-16 between normal and EE patients (25.5 ⁇ 0.4 vs, 26.0 ⁇ 1.1, respectively).
  • Predictive Value Predictive Value Predictive Value miR-146a 0.82 073 0.66
  • MiR-21 gene targeted mice were backcrossed for 5 generations into the C57BL/6 background, described by a previous protocol (Lu, T. et al. J. Immunol. 187:3362-73 (2011)). Littermate controls were used for all experiments. All animals used were housed under specific pathogen-free conditions in accordance with institutional guidelines.
  • Bone marrow cells were collected from femurs and tibia of the mice, and the stem/progenitor cell-enriched low-density fraction was isolated by gradient centrifugation using the Histopaque 1083 (Sigma, St. Louis, MO), according to the manufacturer's protocol.
  • the low density fraction of bone marrow cells were cultured in Iscove's Modified Dulbecco's Media (IMDM) with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 ⁇ streptomycin supplemented with 100 ng/mL stem cell factor and 100 ng/mL FLT-3 ligand (Peprotech, Rocky Hill, NJ) from day 0 to day 4 at a concentration of 1 x 10 6 /mL in 6-well plates.
  • IMDM Iscove's Modified Dulbecco's Media
  • FBS fetal bovine serum
  • penicillin 100 U/ml penicillin
  • streptomycin 100 ng/mL stem cell factor
  • FLT-3 ligand Peprotech, Rocky Hill, NJ
  • the stem cell factor and FLT-3 ligand were replaced with 20 ng/mL granulocyte colony- stimulating factor (G-CSF) on day 4, and the cells were cultured for an additional 6 days in the presence of G-CSF. Eosinophil and neutrophil progenitor growth was assessed by counting the cells every 2 days using a hemacytometer.
  • G-CSF granulocyte colony- stimulating factor
  • the levels of apoptosis in the miR-21 "7" and miR-21 +7+ eosinophil progenitor cultures were measured by Annexin V and 7AAD staining.
  • the viable cells are Annexin V and 7AAD double negative.
  • the early apoptotic cells are Annexin V positive and 7AAD negative.
  • the late apoptotic cells are Annexin V and 7AAD double positive.
  • the miR-21 "7” eosinophil progenitor cultures Compared to miR-21 +7+ cultures, the miR-21 "7" eosinophil progenitor cultures have increased levels of both the Annexin V + 7AAD " population and the Annexin V + 7AAD + population, indicative of increased levels of apoptosis in the miR-21 "7” cultures ( Figure 13).
  • Red blood cells were lysed from mouse blood by using red blood cell (RBC) lysis buffer (Sigma) twice for 5 minutes each time.
  • the eosinophil percentage was determined by FACS staining of blood cells with fluorescein (FITC)-conjugated anti-CCR3 (R&D Systems, Minneapolis, MN) and PE-conjugated anti-Siglec-F (BD Biosciences, San Diego, CA).
  • FITC fluorescein
  • R&D Systems Red Blood Cell
  • PE-conjugated anti-Siglec-F BD Biosciences, San Diego, CA.
  • the eosinophils analyzed were the CCR3 and Siglec-F double positive cells, as described by a previous protocol (Fulkerson, P. et al. Proc. Natl. Acad. Sci. U.S.A. 103: 16418- 16423 (2006)).
  • CFU colony forming unit
  • CFU Colony Forming Unit
  • the Mouse Gene LOST array (Affymetrix) was used to compare gene expression profiles between miR-21 +/+ and miR-2r /_ eosinophil progenitor cultures at day 4, day 8, and day 12.
  • Microarray data were analyzed using GeneSpring software (Agilent Technologies). Global scaling was performed to compare genes from chip to chip, and a base set of probes was generated by requiring a minimum raw expression level of the 20 th percentile out of all probes on the microarray. The resulting probe sets were then baseline transformed and filtered on at least a 1.5 -fold difference between miR-21 +/+ and miR-2r /_ eosinophil progenitor cultures.
  • microarray data have been deposited into the Array Express database, found at http ⁇ colon slash slash> www ⁇ dot> ebi ⁇ dot> ac ⁇ dot> uk ⁇ slash> arrayexpress, with accession number E-MEXP-3346, in compliance with MIAME standards.
  • the up-regulated genes include Ms4a3 and Bhlhal5, each of which is known to have a role in inhibiting cell growth (see, e.g., Donato, J. et al. J. Clin. Invest. 109:51-58 (2002); Jia, D. et al. Gastroenterology 135: 1687-97 (2008)).
  • the down-regulated genes include Grb7 and Hyall, each of which has been shown to promote cell growth (see, e.g., Wang, Y. et al. Clin. Cancer Res. 16:2529-39 (2010); Tan, J. et al. Int. J. Cancer 128: 1303-15 (2011)).
  • MiR-223 gene targeted mice were backcrossed for 5 generations into the C57BL/6 background, as described in a previous protocol (Lu, T. et al. J. Immunol. 187:3362- 73 (2011)). Littermate controls were used for all experiments. All animals used were housed under specific pathogen-free conditions in accordance with institutional guidelines. The Institutional Animal Care and Use Committee of the Cincinnati Children's Hospital Medical Center approved the use of animals in these experiments.
  • Bone marrow cells were collected from femurs and tibia of the mice, and the stem/progenitor cell-enriched low-density fraction was isolated by gradient centrifugation using the Histopaque 1083 (Sigma), according to the manufacturer's protocol.
  • the low density fraction of bone marrow cells were cultured in IMDM with 10% FBS, 100 U/ml penicillin, and 100 g/ml streptomycin supplemented with 100 ng/mL stem cell factor and 100 ng/mL FLT-3 ligand (Peprotech) from day 0 to day 4.
  • IGF1 receptor IGF1 receptor
  • IGFIR was not found to be expressed at day 4, indicating that proliferation of progenitor cells under the influence of stem cell factor and FLT-3 ligand is not dependent on IGFIR levels.
  • Significant levels of IGFIR expression were found from day 10 to day 14 of the culture, coinciding with the increased proliferation seen in both the miR-223 +/+ and miR- 223 _/" eosinophil cultures ( Figure 18A, Figure 19).
  • the IGFIR level progressively decreased from day 10 to day 14, reflecting that eosinophil progenitors gradually lose their proliferation capacity during the differentiation process ( Figure 18 A, Figure 19).
  • the miR-223 "7" cultures have significantly increased levels of IGFIR at both day 12 and day 14 ( Figure 19).
  • Bone marrow-derived eosinophils were re-suspended at a concentration of lxl 0 6 cells/ml and treated with DMSO or 2 ⁇ of picropodophyllin at day 8 (Yin, S. et al. Neuro. Oncol. 12: 19-27 (2010)). Cell growth was determined by cell counting using a hemacytometer on day 10 and day 12. Cell lysates were collected on day 10, and levels of IGF1R expression were determined by western blot.
  • Bone marrow-derived eosinophils were resuspended at a concentration of lxl 0 6 cells/mL and plated in a 96 well plate at 100 [l per well on day 9 to determine a dose response curve of picropodophyllin.
  • the cells were treated with increasing concentrations of picropodophyllin, and the level of cell growth was determined using Cell-Titer Glo luminescent cell viability assay (Promega, Madison, WI), according to the manufacturer's protocol.
  • Eosinophil cultures were treated on day 8 with 2 ⁇ of picropodophyllin, an IGF1R inhibitor, or an equivalent volume of dimethyl sulfoxide (DMSO) as a control. DMSO treatment had no effect on eosinophil proliferation.
  • the Mouse Gene LOST array (Affymetrix) was used to compare gene expression profiles between miR-223 +/+ and miR-223 "7" eosinophil progenitor cultures at day 4, day 8, and day 12.
  • Microarray data were analyzed using GeneSpring software (Agilent Technologies). Global scaling was performed to compare genes from chip to chip, and a base set of probes was generated by requiring a minimum raw expression level of 20 th percentile out of all probes on the microarray. The resulting probe sets were then baseline transformed and filtered on at least a 1.5-fold difference between miR-223 +/+ and miR-223 "7" eosinophil progenitor cultures.
  • microarray data have been deposited into the Array Express database, found at http ⁇ colon slash slash> www ⁇ dot> ebi ⁇ dot> ac ⁇ dot> uk ⁇ slash> arrayexpress, with accession number E-MEXP-3350, in compliance with MIAME standards.
  • Table 8 List of differentially regulated genes between miR-223 and miR-223 " " eosinophil progenitor cultures at day 8.
  • Table 9 List of differentially regulated genes between miR-223 and miR-223 " " eosinophil progenitor cultures at day 12.
  • the EE remission patients responding to steroid treatment had a clinical history of EE, treatment with swallowed topical glucocorticoids, and responsiveness as indicated by an eosinophil count of ⁇ 1 per 400x hpf and normalization of histological features of the disease.
  • the EE remission patients responding to diet treatment had a clinical history of EE, treatment with diet modification, and responsiveness, as described above.
  • the EE patients not responding to glucocorticoid treatment had a clinical history of EE, treatment with swallowed topical glucocorticoid, and non-responsiveness as indicated by an eosinophil count of >24 per 400x hpf.
  • the U937 monocytes and Jurkat T cells were cultured in RPMI 1640 medium (Fisher Scientific) supplemented with 10% FBS, 100 U/mL penicillin, and 100 ⁇ streptomycin.
  • the CCD-16Lu fibroblasts were cultured in Eagle's Minimum Essential Medium (ATCC, Manassas, VA) supplemented with 10% FBS, 100 U/mL penicillin, and 100 ⁇ g/ml streptomycin.
  • RNA was isolated using the miRNeasy Mini Kit (Qiagen), according to the manufacturer's instructions. RNA quality was assessed using the 2100 bioanalyzer (Agilent Technologies), and only samples with RNA integrity number >8 were used. MiRNA expression from human bronchial epithelial cells was profiled using the TaqMan Human MicroRNA Array vl .O (Applied Biosystems), which includes probes for 365 human miRNAs, according to the manufacturer's protocols. Data analysis was carried out using GeneSpring software (Agilent Technologies).
  • the miRNAs differentially regulated between unstimulated and IL-13 stimulated samples were identified by normalizing the expression data to the average of two endogenous control probes, namely RNU44 and RNU48, then filtered on cycle threshold values ⁇ 35 and at least a 2-fold change between unstimulated and IL-13 -stimulated samples. Statistical significance was determined at p ⁇ 0.05 with Benjamini Hochberg false discovery rate correction. The list of differentially expressed miRNAs was clustered using hierarchical clustering, and a heatmap was generated.
  • miRNAs were found to be differentially regulated in response to IL-13 ( Figure 25 A). These include four down-regulated miRNAs, namely miR-375, miR-212, miR-181a-2* (the asterisk indicates the minor form of the miRNA derived from the passenger strand), and miR-145, and 2 up-regulated miRNAs, namely miR-223 and miR-137.
  • IL-13 treated human bronchial epithelial cells found four downregulated miRNAs, namely miR-565, miR-7, miR-335, and miR-375, and 2 up-regulated miRNAs, namely miR-146b and miR-203 (Figure 25B).
  • the study subsequently aimed to investigate the correlation of miR-375 expression with other markers of EE disease activity.
  • the study aimed to determine miR-375 correlation with the high level of eosinophil infiltration observed in the esophageal biopsies of EE patients (see, e.g., Blanchard, C. et al. J. Clin. Invest. 116:536-47 (2006); Abonia, J. et al. J. Allergy Clin. Immunol. 126: 140-9 (2010)) and the expression of previously identified EE signature genes.
  • Esophageal miR-375 expression exhibited a significant inverse correlation with the level of eosinophil infiltration in esophageal biopsies as well as esophageal expression of genes involved in inflammation, including CCL26 (eotaxin-3) (Blanchard, C. et al. J. Clin. Invest. 116:536-47 (2006)), and remodeling, including POSTN (periostin) (Stansfield, W. et al. Ann. Thorac. Surg. 88: 1916-21 (2009)).
  • CCL26 eotaxin-3
  • POSTN perostin
  • Esophageal miR-375 expression also exhibited a significant inverse correlation with the level of esophageal expression of T H 2 cytokines, including IL-5 and IL-13 (Kaiko, G. and Foster, P. Curr. Opin. Allergy Clin. Immunol. 11 :39-45 (2011)) and cell-specific markers for eosinophils (CLC) (Ackerman, S. et al. J. Biol. Chem. 277: 14859-68 (2002)), mast cells (CP A3 and TPSAB1) (Xing, W. et al. Proc. Natl. Acad. Sci. U.S.A. 108:14210-5 (2011)), and epithelial cells (FLG) (Blanchard, C. et al. J. Immunol. 184:4033-41 (2010)) ( Figures 28B-C).
  • CLC cell-specific markers for eosinophils
  • CLC cell-specific markers for eosin
  • Human Genome-Wide mRNA Microarray [ 00295 ] The human Gene LOST array (Affymetrix) was used to compare the gene expression profiles of control-transduced TE-7 cells and pre-miR-375 -transduced TE-7 cells before and after IL-13 treatment. Microarray data were analyzed using GeneSpring software (Agilent Technologies, San Diego, CA), as described in a previous protocol (Lu, T. et al. J. Immunol. 187:3362-73 (2011)).
  • Biological functional enrichment analysis was carried out using Ingenuity Pathway Analysis (Ingenuity Systems) and Toppgene/Toppcluster (Cincinnati Children's Hospital Medical Center) (Chen, J. et al. Nucleic Acids Res. 37:W305- 11 (2009); Kaimal, V. et al. Nucleic Acids Res. 38:W96-102 (2010)).
  • the microarray data have been deposited into the Array Express database, found at http ⁇ colon slash slash> www ⁇ dot> ebi ⁇ dot> ac ⁇ dot> uk ⁇ slash> arrayexpress, with accession number E-MEXP-3345, in compliance with MIAME standards.
  • Esophageal mRNA from EE patients was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems), following the manufacturer's protocol, using the TaqMan reagents (Applied Biosystems) for amplification of EE signature genes (Blanchard, C. et al. J. Allergy Clin. Immunol. 120: 1292-300 (2007); Blanchard, C. and Rothenberg, M. Gastrointest. Endosc. Clin. N. Am. 18: 133-43 (2008); Abonia, J. et al. J. Allergy Clin. Immunol. 126: 140-9 (2010)).
  • Real-time PCR amplification was performed on a TaqMan 7900HT Real-Time PCR System (Applied Biosystems).
  • MiR-375 was able to repress a large set of genes at baseline, consistent with the function of miRNAs as repressors of gene expression (Figure 29 A). A smaller set of genes were induced at baseline; this can be though miR-375 mediated repression of transcriptional repressors (Figure 3 OA). MiR-375 was able to both potentiate and antagonize a subset of IL- 13 mediated gene signatures, indicating a complex interaction between miR-375 and effects of IL-13 ( Figure 30A).
  • Table 11 List of genes differentially regulated by miR-375 in esophageal epithelial cells before and after IL-13 stimulation.
  • Table 12 List of miR-375 -regulated genes that are involved in immunoinflammatory responses.
  • MiR-375 was found to have no effect on TSLP production ( Figure 31), and there was no correlation between miR-375 and TSLP in the esophageal samples.
  • the control- transduced cells and pre-miR-375 -transduced cells expressed TSLP at similar levels without stimulation and have similar levels of induction after polyinosinic:polycytidylic acid (polyLC) stimulation ( Figure 32).
  • Determination of level(s) of miRNAs associated with EE can be used to treat EE. For example, determination of level(s) of miRNAs associated with EE can be used to establish an EE diagnosis, which can then be used to determine an appropriate therapeutic strategy depending on the diagnosis.
  • the treatment method is carried out on a patient to determine the patient's level(s) of miRNAs associated with EE and whether the level of the one or more miRNAs is up-regulated or down-regulated relative to a level of the one or more miRNAs measured in a normal individual, wherein the presence of an elevated or reduced level of one or more miRNAs associated with EE results in the patient being diagnosed with EE.
  • a patient sample is analyzed for expression of at least one of the miRNAs, or a subset of the miRNAs or all of the miRNAs, as listed in Tables 2 and 3.
  • the data is analyzed to determine expression levels of the miRNAs as disclosed herein to establish an EE diagnosis, which is then used to determine an appropriate therapeutic strategy depending on the diagnosis.
  • Determination of level(s) of miRNAs associated with EE can be used to treat EE to determine if a particular drug is or could potentially be effective.
  • the determination of level(s) of miRNAs associated with EE can be used to determine if a therapy specific for a molecule involved in EE disease pathogenesis up- or down-regulates certain EE-associated miRNAs.
  • miR-675 was found to be the only disease remission-induced miRNA, as miR-675 is up-regulated in glucocorticoid responder patients compared to normal, EE, or chronic esophagitis patients. Accordingly, miR-675 can be used to identify, and thereby determine an effective treatment strategy for, glucocorticoid responder patients, as well as EE patients who do not respond to glucocorticoid treatment.
  • Periostin has been demonstrated to have a key role in IL-13 associated remodeling responses. Accordingly, determination of level(s) of miRNAs associated with periostin can be used to identify, and thereby determine an effective treatment strategy for, anti-IL-13 responder patients, as well as EE patients who do not respond to anti-IL-13 treatment.
  • the treatment method is carried out on a patient to determine the patient's level(s) of miRNAs associated with EE and whether the level of the one or more miRNAs is up-regulated or down-regulated relative to a level of the one or more miRNAs measured in a normal individual, wherein the presence of an elevated or reduced level of one or more miRNAs associated with EE results in the patient being diagnosed with EE.
  • a patient sample is analyzed for expression of at least one of the miRNAs, or a subset of the miRNAs or all of the miRNAs, as listed in Tables 2 and 3.
  • the data is analyzed to determine expression levels of the miRNAs as disclosed herein to establish an EE diagnosis, which is then used to determine an appropriate therapeutic strategy depending on the diagnosis.
  • the patient diagnosed with EE is evaluated to determine whether the patient is compliant with and/or exposed to steroid treatment by determining the patient's miR-675 level, wherein an elevated level of miR-675 indicates that the patient is compliant with and/or exposed to steroid treatment.
  • a patient diagnosed with EE for whom steroid therapy has been determined to be the appropriate therapeutic strategy is evaluated following treatment to determine whether the patient is responsive or non-responsive to steroid treatment, wherein an elevated level of miR-675 following treatment indicates that the patient is responsive to steroid treatment.
  • the patient diagnosed with EE is also evaluated to determine whether the patient is likely to be responsive or non-responsive to anti-IL-13 treatment, wherein an elevated level of one or more miRNAs associated with periostin levels, such as miR-223 and/or miR-375, indicates that the patient is likely to be responsive to anti-IL-13 treatment.
  • Determination of level(s) of miRNAs associated with EE can be used to diagnose EE. For example, determination of level(s) of miRNAs associated with EE can be used to establish an EE diagnosis, which can then be used to determine an appropriate therapeutic strategy depending on the diagnosis. [00313] The diagnostic method is carried out on a patient to determine the patient's level(s) of miRNAs associated with EE and whether the level of the one or more miRNAs is up-regulated or down-regulated relative to a level of the one or more miRNAs measured in a normal individual, wherein the presence of an elevated or reduced level of one or more miRNAs associated with EE results in the patient being diagnosed with EE.
  • a patient sample is analyzed for expression of at least one of the miRNAs, or a subset of the miRNAs or all of the miRNAs, as listed in Tables 2 and 3.
  • the data is analyzed to determine expression levels of the miRNAs as disclosed herein to establish an EE diagnosis.
  • Determination of miRNAs level(s), as described herein, can be used to treat eosinophilic disorders other than EE.
  • determination of level(s) of miRNAs associated with an eosinophilic disorder can be used to establish an eosinophilic disorder diagnosis, which can then be used to determine an appropriate therapeutic strategy depending on the diagnosis.
  • the miRNAs associated with EE can also be associated with eosinophilic disorders other than EE, such as EGID and asthma, given that they relate to eosinophil proliferation.
  • eosinophilic disorders other than EE such as EGID and asthma
  • miR-375 has been shown to be involved in asthma. Accordingly, miR-375 can be used as a biomarker alone or in combination with other miRNAs shown to be involved with asthma, such as miR-21 and miR- 223, and the like.
  • the treatment method is carried out on a patient to determine the patient's level(s) of miRNAs associated with an eosinophilic disorder and whether the level of the one or more miRNAs is up-regulated or down-regulated relative to a level of the one or more miRNAs measured in a normal individual, wherein the presence of an elevated or reduced level of one or more miRNAs associated with an eosinophilic disorder results in the patient being diagnosed with an eosinophilic disorder.
  • a patient sample is analyzed for expression of at least one of the miRNAs, or a subset of the miRNAs or all of the miRNAs, as listed in Tables 2 and 3.
  • the numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the application are to be understood as being modified in some instances by the term "about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the application are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable.

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Abstract

L'invention concerne des procédés et des compositions qui concernent, de manière générale, des méthodes de traitement d'œsophagite éosinophile (EE) et de troubles éosinophiles en fournissant ou en favorisant un diagnostic de l'EE et des troubles éosinophiles. En particulier, l'invention concerne l'obtention d'un échantillon provenant d'un patient, puis la quantification dans l'échantillon d'une quantité d'un ou de plusieurs micro-ARN (miARN) associés à l'EE, un niveau modifié du miARN se corrélant à un diagnostic positif de l'EE. Un diagnostic de l'EE peut alors être fourni ou favorisé, sur la base de l'étape de quantification, et un traitement approprié peut être administré au patient. L'invention concerne en outre des nécessaires, des tests et/ou des ensembles de diagnostics qui peuvent être utilisés pour quantifier le ou les miARN associés à l'EE, ainsi que des traitements développés pour réguler à la hausse ou réguler à la baisse un ou plusieurs miARN et/ou leurs voies en aval se rapportant à l'EE ou à l'asthme. L'invention concerne en outre l'utilisation d'inhibiteurs de l'IGF1 et de l'IGF1R pour le traitement de l'EE et de troubles éosinophiles.
PCT/US2013/027503 2012-02-24 2013-02-22 Profils d'expression de micro-arn œsophagiens dans œsophagite éosinophile WO2013126834A1 (fr)

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CA2865154A CA2865154A1 (fr) 2012-02-24 2013-02-22 Profils d'expression de micro-arn ƒsophagiens dans ƒsophagite eosinophile
AU2013222129A AU2013222129B2 (en) 2012-02-24 2013-02-22 Esophageal microRNA expression profiles in eosinophilic esophagitis
US14/380,672 US9260756B2 (en) 2012-02-24 2013-02-22 Esophageal microRNA expression profiles in eosinophilic esophagitis
US14/989,243 US9624545B2 (en) 2012-02-24 2016-01-06 Esophageal microRNA expression profiles in eosinophilic esophagitis

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US10836820B2 (en) 2014-01-10 2020-11-17 Anaptysbio, Inc. Method of treating inflammatory disorder with antibodies directed against interleukin-33 (IL-33)
US10059764B2 (en) 2014-01-10 2018-08-28 Anaptysbio, Inc. Antibodies directed against interleukin-33 (IL-33) and methods of making and using
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US10604577B2 (en) 2015-10-22 2020-03-31 Allakos Inc. Methods and compositions for treating systemic mastocytosis
US11564905B2 (en) 2016-01-13 2023-01-31 Children's Hospital Medical Center Compositions and methods for treating allergic inflammatory conditions
US10544212B2 (en) 2016-04-27 2020-01-28 Pfizer Inc. Anti-IL-33 antibodies, compositions, methods and uses thereof
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US11203638B2 (en) 2017-05-05 2021-12-21 Allakos Inc. Methods and compositions for treating perennial allergic conjunctivitis and keratoconjunctivitis
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